DNA2 inhibitors for cancer treatment

ABSTRACT

Disclosed herein, inter alia, are compositions and methods for inhibiting DNA2.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/866,268filed Jan. 9, 2018, issued as U.S. Pat. No. 10,173,984 which is adivisional of U.S. application Ser. No. 15/428,021 filed Feb. 8, 2017,issued as U.S. Pat. No. 9,932,310, which claims priority to U.S.Application No. 62/292,506 filed Feb. 8, 2016, which is incorporatedherein by reference in entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. CA085344and Grant No. GM078666 awarded by the National Institutes of Health andunder Grant No. W81XWH-09-1-0041 awarded by the United States Army. Thegovernment has certain rights in the invention.

BACKGROUND

DNA replication is the central process of all actively dividing cells.Blocking this process can result in cell cycle arrest, senescence, andapoptosis. Therefore, DNA replication forks are the targets of mostcancer chemotherapeutics. However, a drawback of these therapies is thatthe cancer cell may become resistant to the radiation or chemotherapy.One major conserved DNA repair enzymes is the DNA2 helicase/nuclease(DNA2). Compounds for reducing or inhibiting the repairing functions ofDNA2 remains elusive. Disclosed herein, inter alia, are solutions tothese and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

The disclosure provides a compound, or pharmaceutically acceptable saltthereof, having the formula:

wherein the substituents are as defined herein.

The disclosure provides methods of treating cancer and Fanconi anemiausing the compounds of Formula (I) or (II); methods of treating cancerand Fanconi anemia using the compounds of Formula (I) or (II) inconjunction with chemotherapeutic agents, radiation, or a combinationthereof; methods of sensitizing cancer cells to radiation orchemotherapy using the compounds of Formula (I) or (II) in conjunctionwith radiation or chemotherapeutic agents; methods of inhibiting DNAreplication using the compounds of Formula (I) or (II); methods ofsuppressing DNA double-strand break repair end resection, recombination,over-resection of nascent DNA in cells defective in fork protectionusing the compounds of Formula (I) or (II); restarting stalled DNAreplication forks in cells using the compounds of Formula (I) or (II);and interfering with telomere replication or repair using the compoundsof Formula (I) or (II).

The disclosure provides pharmaceutical compositions containing compoundsof Formula (I) or (II); methods of treating cancer and Fanconi anemiausing these pharmaceutical compositions; methods of treating cancer andFanconi anemia using these pharmaceuticals compositions in conjunctionwith chemotherapeutic agents, radiation, or a combination thereof;methods of sensitizing cancer cells to radiation or chemotherapy usingthese pharmaceutical compositions in conjunction with radiation orchemotherapeutic agents; methods of inhibiting DNA replication usingthese pharmaceutical compositions; methods of suppressing DNAdouble-strand break repair end resection, recombination, over-resectionof nascent DNA in cells defective in fork protection using thesepharmaceutical compositions; restarting stalled DNA replication forks incells using these pharmaceutical compositions; or interfering withtelomere replication or repair using these pharmaceutical compositions.

These and other aspects of the invention are described in more detailherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Three dimensional human DNA2 model and potential pocketsfor screening small molecule DNA2 inhibitors (See also FIGS. 8A-8F andTable 1). FIG. 1A: A homology model for human DNA2 in complex withsingle-stranded DNA (ssDNA). Three potential drug binding pockets arespecified as Sites 1-3. FIG. 1B: Refinement of the DNA2 model structureby molecular dynamics simulation (50 ns). Root mean square deviation(RMSD) values during simulation at three potential drug binding sitesare shown as fluctuating bars. The secondary structures of the sites arerepresented by color, which is specified at the bottom of the graph.FIG. 1C: The linear domain and motif structures and the drug bindingsites of human DNA2. Upper panel: the DNA2 functional domain structure;Middle panel: the three putative drug binding sites; and Bottom panel:the secondary structure motifs. FIG. 1D: Inhibition of DNA2 nucleaseactivity by chemical compounds that were selected from the virtualscreen. Recombinant flag-tagged DNA2 (10 nM) was mixed with ³²P-labeledflap DNA substrates (500 fmol) in the absence or presence of thepotential DNA2 binding chemical compound (250 μM each). The image showsa representative biochemical reaction (37° C., 15 min) that was resolvedusing 15% denaturing polyacrylamide gel electrophoresis (PAGE). Thelocations of substrates and products on the gel are indicated. FIG. 1E:The chemical name and structure of C5.

FIGS. 2A-2G. Inhibitory kinetics of DNA2 nuclease activity, and C5inhibitory effects to ATPase activity and DNA substrate binding capacity(see also FIGS. 9A-9B). The nuclease activity of DNA2 was analyzed inthe presence of varying concentrations of DNA2 enzyme (0.5-5 nM), flapDNA substrate (5-50 nM), and DNA2 inhibitors C5 (0-250 μM). FIG. 2A:Lineweaver-Burk plot of DNA2 nuclease activity in the presence ofvarious concentrations of flap DNA substrate (x axis) and C5 (μM,designated as [I]). FIG. 2B: DNA2 nuclease activity in the presence ofvarious concentrations of compound C5 (x axis) and flap DNA substrate(nM, designated as [S]). The C5 concentration (IC50_(observed)) thatinhibits 50% of the DNA2 nuclease activity at a given concentration ofDNA substrates is indicated with dotted lines. The inhibited nucleaseactivities were normalized to the DMSO control, set as 1. FIG. 2C: Plotof IC50_(observed) versus [S] for determination of IC50. The values ofIC50_(observed) obtained from panel B and corresponding DNA substrateconcentrations were plotted. When [S] is zero, the derived correspondingIC50_(observed) value is the theoretical IC50 of C5. In FIGS. 2A-2C, thevalues are means±s.d of three independent experiments. FIG. 2D: Therepresentative TLC image showing C5 inhibition of the ATPase activity ofDNA2. The DNA2 enzyme concentration used was 10 nM; ATP substrateconcentration used was 200 μM; DNA concentration used was 200 nM; Theinhibitor C5 concentrations used was in a range of 0 to 250 M. FIG. 2E:Quantification of inhibition of DNA2 in the ATPase activity, therelative ATPase activities normalized to DMSO control. The values shownare the means±s.d. of three independent assays. FIG. 2F: Therepresentative EMSA image showing C5 inhibition of DNA2 binding to theDNA substrate. The DNA2 enzyme concentration used was 50 nM; the ³²Plabeled DNA concentration used was 1 nM; the compound C5 concentrationsused ranged from 0 to 1,000 μM. FIG. 2G: Quantification of inhibition ofDNA2 substrate binding, the relative binding activities normalized toDMSO control. The values shown are the means±s.d. of three independentassays.

FIGS. 3A-3C. DNA2 mutations at Site 1 impair C5 inhibition of DNA2nuclease activity (see also FIGS. 10A-10C). FIG. 3A: Three dimensionalstructure of the Site 1 small molecule binding pocket of DNA2. The leftpanel shows a cartoon view and the right panel shows a surface view ofSite 1. FIG. 3B: The 14 residues within 6 Å spheres around compound C5that form Site 1 pocket were identified. Among them, F696A and L732A,which did not affect the nuclease activity of DNA2 (FIGS. 11A-11B),reduced C5 inhibition of DNA2 nuclease activity. The nuclease activityof WT, F696A, and L732A (1 nM) was assayed in the presence of variousconcentrations of C5 (indicated as [I] in a range from 0 to 250 μM) andquantified, the DNA substrate concentration was 15 nM. We added DMSOwithout the inhibitor C5 as a control where the relative nucleaseactivity was set as 1. The values shown are the means±s.d. of threeindependent experiments. FIG. 3C: The DNA binding activity of F696A andL732A is resistant to C5 inhibitor. We added DMSO without the inhibitorC5 as a control where the relative binding activity was set as 1. TheDNA2 enzyme concentration used was 50 nM. The DNA concentration used was1 nM. The inhibitor C5 concentrations ranged from 0 to 125 μM. Thevalues shown are the means±s.d. of three independent experiments.

FIGS. 4A-4C. IC50 and on-target cytotoxic effects of C5 in human cancercells and mouse embryonic stem (MES) cells. FIG. 4A: IC50 values of C5with a panel of 18 cell lines from 4 major types of cancers. Humannon-cancerous or cancer cells were seeded on a 96-well plate andincubated in culture medium containing 0 to 80 μM C5 for 7 days. TheIC50 was calculated using the CompuSyn software (Chou, 2010). Values arethe average of two independent assays. FIG. 4B: Control (shSCR) or DNA2knockdown MCF7 cells were cultured in medium containing 0 or 1 μM C5 for4 days. The live cells were counted. The cell survival was calculated bynormalizing the number of live cells from each culture to that of thecontrol MCF7 cells (shSCR), which was arbitrarily set as 100. FIG. 4C:The same experiment as in A was performed on MES cells from WT and DNA2knockout mice, which were cultured in medium containing 0 or 1 μM C5 for4 days. The values shown are the means±s.d. of three experiments.

FIGS. 5A-5H. Inhibitor C5 suppresses resection-related homology directedrepair (HDR) and single strand annealing (SSA) and causes accumulationof phosphorylated RPA foci (see also FIGS. 11A-11B). FIG. 5A: C5inhibits HDR and SSA frequency. The U2OS cells carrying the GFP reportergene for HDR or single-strand annealing (SSA) assay were transfectedwith I-Sce I expression vector. The cells were then incubated in mediumcontaining 0, 10, 20, 40, 60 μM C5. After 72 h, the cells were harvestedand the GFP positive cells were analyzed by flow cytometry. In the DNA2knockdown experiment, the U2OS cells were transfected with 10 nM ofscrambled or DNA2 siRNA oligos for 24 hours. The cells were thentransfected with the I-SceI expression vector. After 48 h, the cellswere harvested and the GFP positive cells were analyzed by flowcytometry. Knockdown of DNA2 in the engineered U2OS cells was confirmedby western blot (FIG. 11A-11B). Values are mean±s.d. of threeindependent experiments. FIG. 5B: DNA2 inhibition by siRNA or C5 impairsreplication fork-related DNA end resection in MCF7 cells at similarlevels. MCF7 cells were untreated or treated with 10 μM C5 for 24 hours(left panels) or treated with scrambled siRNA (siControl) or siRNAagainst DNA2 (siDNA2) for 72 hours (right panels). The knockdownefficiency of DNA2 was checked by western blotting. The cells were thentreated with 1 μM CPT for 4 hours. The levels of γ-H2AX andphosphorylated RPA (S33) were analyzed by western blot using antibodiesagainst γH2AX (Millipore) and phosphorylated RPA (S33) (Abcam). Totallevel of RPA and β-actin were used as controls, which were detectedusing antibodies against RPA32 (Abcam) and β-actin (GeneTex). FIGS.5C-5H: DNA2 inhibition by siRNA or C5 impairs replication fork-relatedDNA end resection in A549 cells at similar levels. A549 cells wereuntreated or treated with 10 μM C5 for 24 hours (FIGS. 5C-5E) or treatedwith scrambled siRNA (siControl) or siRNA against DNA2 (siDNA2) for 48hours (FIGS. 5F-5H). The knockdown efficiency of DNA2 was checked bywestern blotting (FIGS. 11A-11B). The cells were then treated with 1 μMCPT for 4 hours. FIGS. 5C and 5F: Representative images. FIGS. 5D, 5E,5G, and 5H: Quantifications: the levels of γ-H2AX and phosphorylated RPA(S33) were quantified by ImagePro Premier, and the relative P-RPA orγH2AX per nucleus was calculated. Values are means±s.d. of threeindependent assays.

FIGS. 6A-6D. C5 suppresses restart of stalled DNA replication forks andover-resection of nascent DNA in cells defective in fork protection (seealso FIGS. 12A-12B). FIGS. 6A and 6B: C5 inhibits DNA replication forkrestart at similar levels as DNA2 knockdown. FIG. 6A: A549 cells weremock or pretreated with C5 (20 μM) for 2 hours, labeled with IdU (red)for 30 min, and co-cultured with IdU and the indicated drugs (20 μM C5,150 nM CPT or 2 mM HU) or drug combinations (20 μM C5 combined with 150nM CPT or 2 mM HU) for 1 hour, and then washed and labeled with CIdU(green) for 40 min. Percentage of restarting forks (red-green tracks)was calculated by dividing the red-green tracks by the sum of thered-green and red only tracks. At least 150 tracks counted for eachsample as shown in left panels were calculated. Values are means±s.d.from three independent experiments (Right panel). The p value wascalculated by the student's t-test. FIG. 6B: For the control experimentin which knockdown of DNA2 was employed, A549 cells were transfectedwith scrambled or DNA2 siRNAs for 72 hours, and the ability of the cellsto restart replication after CPT or HU fork stalling was determined asin panel A. Western blotting confirmed an efficient knockdown of DNA2 at72h post siRNA transfections (FIG. 11A-11B). In both panels A and B, redtracks represent synthesis before addition of HU or CPT. Red/greentracks represent molecules that recovered from fork stalling. Green onlytracks represent initiations after removal of HU or CPT. FIGS. 6C-6D. C5prevents single-stranded DNA accumulation in BOD1L-depleted orBRCA2-depleted U2OS cells upon replication stalling with HU at similarlevel as DNA2 knockdown. FIG. 6C: Cells with more than 15 P-RPA fociindicative of single-stranded DNA were scored in U2OS cells transfectedwith siRNA against BRCA2 or BOD1L or scrambled siRNA, as indicated,followed by treatment with 4 mM hydroxyurea (HU) for 5 h. Cells werepretreated with MRE11 inhibitor mirin (50 μM) or DNA2 inhibitor C5 (20μM). In the absence of HU, no cells with greater than 15 foci wereobserved. FIG. 6D: Cells with more than 15 P-RPA foci indicative ofsingle-stranded DNA were scored in U2OS cells transfected with siRNAagainst BRCA2 or scrambled siRNA, as indicated, followed by treatmentwith 4 mM HU for 5 h. Cells were pretreated with MRE11 inhibitor, PFM39or siRNA against DNA2. In both FIGS. 6C and 6D, top panels show therepresentative immunofluorescence images, and the bottom panel shows thequantifications. Error bars represent the SEM. p-values were calculatedwith the student T-test. Western blots of knockdowns are shown in FIGS.12A-12B.

FIGS. 7A-7C. DNA2 inhibitor C5 synergistically kills breast cancer cellsMCF7 with CPT and PARP inhibitor MK4827. FIG. 7A: C5 sensitizes MCFcells to CPT. Clonogenic assays were conducted to evaluate the survivalrate of MCF cells treated with different concentrations of CPT in theabsence or presence of C5 (1 μM). The survival rate of the cells treatedwith various concentrations of CPT was calculated by normalizing thenumber of colonies to that of the cells without CPT treatment. Thesurvival rate of the cells without CPT treatment was arbitrarily setas 1. The values shown are the means±s. d. of three independentexperiments. FIGS. 7B-7C: The synergy between the DNA2 inhibitor C5 andthe PARP inhibitor MK4827 was assayed by clonogenic assay. The valuesare means±s.d. of three independent clonogenic assays. The IC50 andcombination index (CI) was calculated using the Compusyn program. FIG.7B: Representative inhibition curve of varying concentrations of C5 from0 to 10 μM in combination with MK4827 (0 or 1 μM). FIG. 7C:Representative inhibition curve of varying concentrations of MK4827 from0 to 1 μM in combination with C5 (0 or 2 μM).

FIGS. 8A-8F. FIG. 8A and FIG. 8B shows elimination of DNA2 genesensitizes mouse ES (MES) cells to radiation and camptothecin (CPT) FIG.8A: The survival curves of WT and dna2^(−/−) MES cells after ionizingradiation (IR) treatment. WT and dna2^(−/−) MES cells were exposed to 1,2, 4, 6 gray (Gy) γ-irradiation. The surviving MES cell colonies werecounted and normalized to the corresponding untreated control, whosesurvival rate was arbitrarily set as 1. FIG. 8B: The survival curve ofthe WT and dna2^(−/−) MES cells after CPT treatment. The WT anddna2^(−/−) MES cells were cultured in the medium containing 6.25, 12.5,25, and 100 nM CPT. The surviving ES cell colonies were counted andnormalized to corresponding untreated control, whose survival rate wasarbitrarily set as 1. In both panels, each point represents the mean±s.dof three independent assays. FIGS. 8C-8F show DNA2, FEN1 and EXO1nuclease assays used to test the inhibitor specificity. FIGS. 8C-E showDNA2, FEN1, and EXO1 nuclease assays with the inhibitor titration,respectively. FIG. 8F shows quantification of DNA2 FEN1 and EXO1activities. The enzyme concentrations used for DNA2, FEN1 and EXO1 areall 10 nM. The DNA substrate concentration used was 50 nM. A range ofthe inhibitor C5 concentrations used was from 0 to 125 μM. The relativeactivities normalize to DMSO, which activity set as 1. Each experimentwas repeated at least three times. The error bar represents the standarddeviations. Symbols used: (diamond) FEN1, (square) EXO1, (triangle)DNA2.

FIGS. 9A-9B. (see also FIGS. 2A-2G): FIG. 9A. Time course of flapsubstrate cleavage catalyzed by DNA2 enzyme. Various DNA2 concentrationsand various substrate concentrations were used to determine the propertime for assays. 12 time points were tested. The efficiency is the bestduring 1-10 minutes. FIG. 9B: Inhibition of DNA2 helicase activity byC5. hDNA2 D277A, nuclease-deficient, was purified as describedpreviously. The helicase substrate is a 43 base oligonucleotide annealedto M13DNA over 24 bases and having an 18 nt 5′ noncomplementary tail.The oligonucleotide was 5′ labeled with [g-32P] ATP by T4 polynucleotidekinase and purified with a Sepharose CL4B column. The standard reactionmixture contained 50 mM Tris-HCl, pH7.5, 2 mM DTT, 0.25 mg/ml BSA, 4 mMMgCl₂, 4 mM ATP and 32P-labeled helicase substrate. C5 was preincubatedwith enzyme for 1 min. The reaction was for 30 min at 37° C. Thereaction was stopped with 5× stop solution (60 mM EDTA, 40% sucrose,0.6% SDS, 0.25% bromophenol blue and 0.25% zylene cyanole FF). Reactionproducts were separated on an 8% native polyacrylamide gel containing0.1% SDS and detected by phosphorimaging and quantified usingImageQuant. Lane 1, boiled substrate; Lane 2, no enzyme, Lanes 3-6contained 5, 10 and 15 nM DNA2, respectively; lanes 7-10 contained 5,10, and 15 nM DNA2 plus 100 mM C5. Lanes 3, 4 and 5 represent the amountof helicase at each of the three increasing helicase concentrations inthe absence of C5. Lanes 6, 7, and 8 represent the same amount of enzymeas 3, 4, 5, respectively, but in the presence of C5. The percent ofhelicase remaining after C5 treatment is presented as the amount ofsubstrate unwound in lane 6 divided by the amount in lane 3, the amountof substrate unwound in lane 7 divided by the amount in lane 4, and theamount of substrate unwound in lane 8 divided by the amount in lane 5.

FIGS. 10A-10C. 14 residues in Site 1 were mutated to determine the DNA2binding capacity to the inhibitor C5 (See also FIGS. 3A-3C). FIG. 10A:Nuclease assay to determine the activities of the WT and mutants. Theenzyme concentrations used for DNA2 and its mutants are all 10 nM. TheDNA substrate concentration used was 50 nM. FIG. 10B: Quantification ofthe WT and mutants nuclease activities. The WT activity was set as 1.FIG. 10C: Nuclease activity assays to measure the sensitivity of the WT(lane 1-9) and mutants F696A (lanes 10-18) and L732A (lanes 19-27) tothe inhibitor C5. The enzyme concentrations used for DNA2 and itsmutants are all 10 nM. The DNA substrate concentration used was 50 nM.The inhibitor concentrations used were in a range from 0 to 1000 μM.

FIGS. 11A-11B. FIG. 11A: Cell cycle analysis by flow cytometry. HCT116cells were treated with 0, 10, 50 μM C5 for 48 h. The cells were stainedby PI and the cell cycle profile was determined by flow cytometry andcalculated by the Flow Jo software based on DNA contents. FIG. 11B:Knockdown of DNA2 in MCF7, A549, and U2OS cells. Cells at 50-60%confluence were transfected with scramble siRNA oligos (siControl) orsiRNA oligos against human DNA2 (Sigma). After 48 hours incubation,cells were harvested and lysed. The level of DNA2 were detected bywestern blot using an antibody against human DNA2. The level of β-actinwas used as a loading control. Antibodies were from Abgent.

FIGS. 12A-12B. (Also see FIGS. 5A-5H). FIG. 12A: DNA fiber assayconfirms that C5 inhibits DNA replication fork restart in the presenceof high concentration of CPT. A549 cells were first labeled with IdU(red) and co-cultured with the indicated drugs (10 μM C5 or 2 μM CPT) ordrug combinations (10 μM C5 and 2 μM CPT), and then washed and labeledwith CIdU (green). The different types of typical tracks. Left panels:representative images of different tracks; right panel: Quantificationof the different tracks found in the cells with different treatments.Approximate 150 tracks were scored in each assay. Values are means±s.d.from three independent experiments. The p value was calculated with thestudent's t-test. FIG. 12B: Knockdown of DNA2, BRCA2, and BOD1L in U2OScells. The cells at 50-60% confluence were transfected with scramblesiRNA oligos or siRNA oligos (Sigma) against human BOD1L, BRCA2 and DNA2individually or in combination. The levels of BOD1L, BRCA2, or DNA2 weredetected by western blot using an antibody against human BOD1L, BRCA2,or DNA2. The level of β-actin was used as a loading control. Antibodieswere from Abgent.

FIGS. 13A-13C. Synergy between C5 and FEN1. EXO1 or MRE11 knockdownindicates C5 has on-target effects in vivo in human cancer cells. On day1, HCT116 DNA2^(flox/+/+) and HCT116 DNA2^(flox/−/−) cells (3*10⁶ wereplated in 10 cm dishes, respectively. On day 2, the genetic knockout ofDNA2 was induced with tamoxifen as previously described (Karanja et al.,2014). Control wells contained EtOH. On day 3, the tamoxifen floxedcells, now DNA2^(−/+/+) and DNA2^(−/−/−), and DNA2^(flox/+/+) controlswere plated on 24 well plates, 20,000 to 30,000 cells per well, andincubated overnight. On day 4, wells were transfected with 20 pmolecontrol SCR siRNA or FEN1 (FIG. 13A), EXO1 (FIG. 13B), or MRE11 (FIG.13C) si RNAs, respectively, as previously described using GeneMute.siRNAs were obtained from Invitrogen: FEN1 (HSS103627, HSS103629,HSS176903), MRE11 (HSS142960, HSS142961, HSS181171), EXO1 (HSS113557,HSS113558, HSS113559). Cells were incubated overnight. To test ifinhibition of DNA2 by C5 showed synthetic lethality with FEN1, EXO1 orMre11 si RNA knockdown, C5 was added to each well of DNA2^(flox/+/+), asindicated (compare red and blue curves). Cells were cultured for anadditional 3 days, until DNA2^(flox/+/+) cells lacking C5 wereconfluent. As a control for the efficacy of C5, the effect of geneticknockdown of DNA2 was tested in the presence the respective siRNAtreated DNA2^(−/−/−) (gray curves). Cells were then washed with PBS,trypsinized and stained with tryphan blue to differentiate live cellsfrom dead cells. Cells were counted microscopically in a Neubauerchamber. Error bars represent data from duplicate experiments. Bothinhibition of DNA2 by C5 and genetic knockout of DNA2 with tamoxifenproduces synthetic lethality with FEN1, EXO1 or MRE11 deficiency.

FIG. 14. Yeast cells with the designated genotypes were treated with theindicated levels of C5 and a derivative, C5-2, and survival aftertreating with the DNA damaging agent methyl methane sulfonate orincubating at high temperature was determined by dilution assay.Colonies represent serial 10 fold dilutions of the yeast strains. Thetwo rows labeled rad27 are duplicate experiments. C5 suppressescisplatin sensitivity in FANCD2−/− fibroblasts. We previously showedthat knockdown of DNA2 suppressed the cisplatin sensitivity of FANCD2−/−fibroblasts. This suggests that C5 should also suppress the cisplatinsensitivity of FANCD2−/− cells, which might protect non-cancerous cellsin FA patients from treatment of tumors with cisplatin.

FIG. 15. Cells were treated with cisplatin in the presence of increasingamounts of C5 or in the absence of C5. The experiment was performed induplicate. As shown in FIG. 15, at 125 nM cisplatin, cells treated with10 μM C5 showed an increase in survival compared to cells not treatedwith C5.

DETAILED DESCRIPTION

Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl,n-heptyl, n-octyl, and the like. An unsaturated alkyl group is onehaving one or more double bonds or triple bonds. Examples of unsaturatedalkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. An alkoxy is an alkyl attached to the remainder of the moleculevia an oxygen linker (—O—). An alkyl moiety may be an alkenyl moiety. Analkyl moiety may be an alkynyl moiety. An alkyl moiety may be fullysaturated. An alkenyl may include more than one double bond and/or oneor more triple bonds in addition to the one or more double bonds. Analkynyl may include more than one triple bond and/or one or more doublebonds in addition to the one or more triple bonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched non-cyclicchain, or combinations thereof, including at least one carbon atom andat least one heteroatom (e.g. O, N, P, Si, and S), and wherein thenitrogen and sulfur atoms may optionally be oxidized, and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) (e.g. O, N,P, S, and Si) may be placed at any interior position of the heteroalkylgroup or at the position at which the alkyl group is attached to theremainder of the molecule. Examples include, but are not limited to:—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up totwo or three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include oneheteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includetwo optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include three optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include fouroptionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P). The term“heteroalkenyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one doublebond. A heteroalkenyl may optionally include more than one double bondand/or one or more triple bonds in additional to the one or more doublebonds. The term “heteroalkynyl,” by itself or in combination withanother term, means, unless otherwise stated, a heteroalkyl including atleast one triple bond. A heteroalkynyl may optionally include more thanone triple bond and/or one or more double bonds in additional to the oneor more triple bonds.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated,non-aromatic cyclic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,3-hydroxy-cyclobut-3-enyl-1,2, dione, 1H-1,2,4-triazolyl-5(4H)-one,4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include,but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A“cycloalkylene” and a “heterocycloalkylene,” alone or as part of anothersubstituent, means a divalent radical derived from a cycloalkyl andheterocycloalkyl, respectively. A heterocycloalkyl moiety may includeone ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moietymay include two optionally different ring heteroatoms (e.g., O, N, S,Si, or P). A heterocycloalkyl moiety may include three optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include four optionally different ring heteroatoms (e.g., O,N, S, Si, or P). A heterocycloalkyl moiety may include five optionallydifferent ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkylmoiety may include up to 8 optionally different ring heteroatoms (e.g.,O, N, S, Si, or P).

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. Non-limiting examples of aryl and heteroarylgroups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl,indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl,pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl,benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl,pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl,benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl,diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl,pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl,or quinolyl. The examples above may be substituted or unsubstituted anddivalent radicals of each heteroaryl example above are non-limitingexamples of heteroarylene. A heteroaryl moiety may include one ringheteroatom (e.g., O, N, or S). A heteroaryl moiety may include twooptionally different ring heteroatoms (e.g., O, N, or S). A heteroarylmoiety may include three optionally different ring heteroatoms (e.g., O,N, or S). A heteroaryl moiety may include four optionally different ringheteroatoms (e.g., O, N, or S). A heteroaryl moiety may include fiveoptionally different ring heteroatoms (e.g., O, N, or S). An aryl moietymay have a single ring. An aryl moiety may have two optionally differentrings. An aryl moiety may have three optionally different rings. An arylmoiety may have four optionally different rings. A heteroaryl moiety mayhave one ring. A heteroaryl moiety may have two optionally differentrings. A heteroaryl moiety may have three optionally different rings. Aheteroaryl moiety may have four optionally different rings. A heteroarylmoiety may have five optionally different rings.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl”, “heteroalkyl”, “cycloalkyl”,“heterocycloalkyl”, “aryl”, and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R′″, and R″″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″, and R″″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″ R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′ R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system; and where R′, R″,R′″, and R″″ are preferably independently selected from hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupswhen more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂) r-B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties: (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,—NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, substituted with at least onesubstituent selected from: (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, substituted with at least onesubstituent selected from: (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, substituted with at least onesubstituent selected from: oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,—NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present invention. Salts tend to be more soluble inaqueous or other protonic solvents than are the corresponding free baseforms. In other cases, the preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Provided herein are agents (e.g. compounds, drugs, therapeutic agents)that may be in a prodrug form. Prodrugs of the compounds describedherein are those compounds that readily undergo chemical changes underselect physiological conditions to provide the final agents (e.g.compounds, drugs, therapeutic agents). Additionally, prodrugs can beconverted to agents (e.g. compounds, drugs, therapeutic agents) bychemical or biochemical methods in an ex vivo environment. Prodrugsdescribed herein include compounds that readily undergo chemical changesunder select physiological conditions to provide agents (e.g. compounds,drugs, therapeutic agents) to a biological system (e.g. in a subject).

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers. In embodiments, the compounds described herein are tautomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The terms “treating” or “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, disease, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,certain methods herein treat hyperproliferative disorders, such ascancer (e.g. ovarian cancer, bladder cancer, head and neck cancer, braincancer, breast cancer, lung cancer, cervical cancer, bone cancer, spinalcancer, liver cancer, colorectal cancer, pancreatic cancer,glioblastoma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, renalcancer, renal cell carcinoma, non-small cell lung cancer, uterinecancer, testicular cancer, anal cancer, bile duct cancer, biliary tractcancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, appendix cancer, small intestine cancer, stomach(gastric) cancer, urinary bladder cancer, genitourinary tract cancer,endometrial cancer, nasopharyngeal cancer, head and neck squamous cellcarcinoma, or prostate cancer). For example certain methods herein treatcancer by decreasing or reducing or preventing the occurrence, growth,metastasis, or progression of cancer or by decreasing or reducing orpreventing a symptom of cancer. Symptoms of cancer (e.g., ovariancancer, bladder cancer, head and neck cancer, brain cancer, breastcancer, lung cancer, cervical cancer, bone cancer, spinal cancer, livercancer, colorectal cancer, pancreatic cancer, glioblastoma,neuroblastoma, rhabdomyosarcoma, osteosarcoma, renal cancer, renal cellcarcinoma, non-small cell lung cancer, uterine cancer, testicularcancer, anal cancer, bile duct cancer, biliary tract cancer,gastrointestinal carcinoid tumors, esophageal cancer, gall bladdercancer, appendix cancer, small intestine cancer, stomach (gastric)cancer, urinary bladder cancer, genitourinary tract cancer, endometrialcancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, orprostate cancer) would be known or may be determined by a person ofordinary skill in the art. The term “treating” and conjugations thereof,include prevention of an injury, pathology, condition, or disease (e.g.preventing the development of one or more symptoms of cancer (e.g.ovarian cancer, bladder cancer, head and neck cancer, brain cancer,breast cancer, lung cancer, cervical cancer, bone cancer, spinal cancer,liver cancer, colorectal cancer, pancreatic cancer, glioblastoma,neuroblastoma, rhabdomyosarcoma, osteosarcoma, renal cancer, renal cellcarcinoma, non-small cell lung cancer, uterine cancer, testicularcancer, anal cancer, bile duct cancer, biliary tract cancer,gastrointestinal carcinoid tumors, esophageal cancer, gall bladdercancer, appendix cancer, small intestine cancer, stomach (gastric)cancer, urinary bladder cancer, genitourinary tract cancer, endometrialcancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, orprostate cancer).

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, increase enzyme activity, reduceprotein function, reduce one or more symptoms of a disease orcondition). An example of an “effective amount” is an amount sufficientto contribute to the treatment, prevention, or reduction of a symptom orsymptoms of a disease, which could also be referred to as a“therapeutically effective amount.” A “reduction” of a symptom orsymptoms (and grammatical equivalents of this phrase) means decreasingof the severity or frequency of the symptom(s), or elimination of thesymptom(s). A “prophylactically effective amount” of a drug or prodrugis an amount of a drug or prodrug that, when administered to a subject,will have the intended prophylactic effect, e.g., preventing or delayingthe onset (or reoccurrence) of an injury, disease, pathology orcondition, or reducing the likelihood of the onset (or reoccurrence) ofan injury, disease, pathology, or condition, or their symptoms. The fullprophylactic effect does not necessarily occur by administration of onedose, and may occur only after administration of a series of doses.Thus, a prophylactically effective amount may be administered in one ormore administrations. The exact amounts will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (such ascancer) means that the disease is caused by (in whole or in part), or asymptom of the disease is caused by (in whole or in part) the substanceor substance activity or function. As used herein, what is described asbeing associated with a disease, if a causative agent, could be a targetfor treatment of the disease. For example cancer may be treated with acomposition (e.g. compound, composition, nanoparticle, or conjugate, allas described herein) effective for inhibiting DNA replication.

“Control” or “control experiment” or “standard control” is used inaccordance with its plain ordinary meaning and refers to an experimentin which the subjects or reagents of the experiment are treated as in aparallel experiment except for omission of a procedure, reagent, orvariable of the experiment. In some instances, the control is used as astandard of comparison in evaluating experimental effects.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules, or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. The term “contacting” may includeallowing two species to react, interact, or physically touch, whereinthe two species may be a compound as described herein and a protein orenzyme. In some embodiments contacting includes allowing a compounddescribed herein to interact with a protein. In some embodimentscontacting includes allowing a compound described herein to interactwith a stromal cell. In some embodiments contacting includes allowing acompound described herein to interact with an immune cell. In someembodiments contacting includes allowing a compound described herein tointeract with a protein associate with a stromal cell. In someembodiments contacting includes allowing a compound described herein tointeract with a protein associated with an immune cell. In someembodiments contacting includes allowing a compound described herein tointeract with the extracellular matrix generated by a stromal cell. Insome embodiments contacting includes allowing a compound describedherein to interact with the extracellular matrix generated by an immunecell.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor (e.g. antagonist)interaction means negatively affecting (e.g. decreasing) the level ofactivity or function of the protein relative to the level of activity orfunction of the protein in the absence of the inhibitor. In embodiments,inhibition refers to a decrease in DNA replication or transcription. Insome embodiments inhibition refers to reduction of a disease or symptomsof disease, such as cancer. Thus, inhibition may include, at least inpart, partially or totally blocking stimulation, decreasing, preventing,or delaying activation, or inactivating, desensitizing, ordown-regulating signal transduction or enzymatic activity or the amountof a protein.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein-activator (e.g. agonist) interactionmeans positively affecting (e.g. increasing) the activity or function ofthe protein relative to the activity or function of the protein in theabsence of the activator (e.g. compound described herein). Thus,activation may include, at least in part, partially or totallyincreasing stimulation, increasing or enabling activation, oractivating, sensitizing, or up-regulating signal transduction orenzymatic activity or the amount of a protein decreased in a disease.Activation may include, at least in part, partially or totallyincreasing stimulation, increasing or enabling activation, oractivating, sensitizing, or up-regulating signal transduction orenzymatic activity or the amount of a protein.

The term “modulator” refers to a composition that increases or decreasesthe level of a target molecule or the function of a target molecule.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemia,lymphoma, carcinomas and sarcomas. Exemplary cancers that may be treatedwith a compound, pharmaceutical composition, or method provided hereininclude ovarian cancer, lymphoma, sarcoma, bladder cancer, bone cancer,brain tumor, cervical cancer, colon cancer, esophageal cancer, gastriccancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer,leukemia, prostate cancer, breast cancer (e.g. ER positive, ER negative,chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicinresistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma,primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer(e.g. hepatocellular carcinoma), lung cancer (e.g. non-small cell lungcarcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lungcarcinoma, small cell lung carcinoma, carcinoid, sarcoma, cisplatinresistant lung cancer, carboplatin resistant lung cancer, platinum-basedcompound resistant lung cancer), glioblastoma multiforme, glioma, ormelanoma. Additional examples include, cancer of the thyroid, endocrinesystem, brain, breast, cervix, colon, head & neck, liver, kidney, lung,non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach,uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme,ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, primary brain tumors, cancer, malignant pancreaticinsulanoma, malignant carcinoid, urinary bladder cancer, premalignantskin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms ofthe endocrine or exocrine pancreas, medullary thyroid cancer, medullarythyroid carcinoma, melanoma, colorectal cancer, papillary thyroidcancer, hepatocellular carcinoma, Paget's Disease of the Nipple,Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of thepancreatic stellate cells, cancer of the hepatic stellate cells, orprostate cancer. In embodiments “cancer” refers to a cancer resistant toan anti-cancer therapy (e.g. treatment with an anti-cancer agent). Inembodiments, the cancer is breast cancer. In embodiments, the cancer iscolon cancer. In embodiments, the cancer is prostate cancer. Inembodiments, the cancer is lung cancer. In embodiments, the cancer isovarian cancer.

“Radiation” and “radiation therapy” refers to any in the art, andincludes external beam radiation therapy, internal radiation therapy,and systemic radiation therapy. Systemic radiation therapy refers toadministering a patient a radiopharmaceutical (e.g., radioactivesubstances such as iodine, strontium, samarium, radium; and radioactivesubstances bound to monoclonal antibodies).

“Patient” or “subject in need thereof” or “subject” refers to a livingorganism suffering from or prone to a disease or condition that can betreated by administration of a compound or pharmaceutical composition orby a method, as provided herein. Non-limiting examples include humans,other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows,deer, and other non-mammalian animals. In some embodiments, a patient ishuman. In some embodiments, a subject is human.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is cancer.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intracranial, intranasal or subcutaneous administration, or theimplantation of a slow-release device, e.g., a mini-osmotic pump, to asubject. Administration is by any route, including parenteral andtransmucosal (e.g., buccal, sublingual, palatal, gingival, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc. By“co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional compositions (e.g.,chemotherapeutic agents, radiopharmaceuticals) or therapies (e.g.,radiation). The compound of the invention can be administered alone orcan be coadministered with a chemotherapeutic agent and/or aradiopharmaceutical. Coadministration is meant to include simultaneousor sequential administration of the compound individually or incombination with one or more chemotherapeutic agents and/or one or moreradiopharmaceuticals. Thus, the preparations can also be combined, whendesired, with other active substances (e.g. to reduce metabolicdegradation, to increase degradation of a prodrug and release of thedrug, detectable agent). The compositions of the present invention canbe delivered by transdermally, by a topical route, formulated asapplicator sticks, solutions, suspensions, emulsions, gels, creams,ointments, pastes, jellies, paints, powders, and aerosols. Oralpreparations include tablets, pills, powder, dragees, capsules, liquids,lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. Liquid form preparations include solutions, suspensions, andemulsions, for example, water or water/propylene glycol solutions. Thecompositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes. The compositions of the present invention can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). In another embodiment, theformulations of the compositions of the present invention can bedelivered by the use of liposomes which fuse with the cellular membraneor are endocytosed, i.e., by employing receptor ligands attached to theliposome, that bind to surface membrane protein receptors of the cellresulting in endocytosis. By using liposomes, particularly where theliposome surface carries receptor ligands specific for target cells, orare otherwise preferentially directed to a specific organ, one can focusthe delivery of the compositions of the present invention into thetarget cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul.13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro,Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions of the presentinvention can also be delivered as nanoparticles.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient (e.g. compounds describedherein, including embodiments or examples) may be contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. When administered in methods to treat a disease, suchcompositions will contain an amount of active ingredient effective toachieve the desired result, e.g., reducing, eliminating, or slowing theprogression of disease symptoms. Determination of a therapeuticallyeffective amount of a compound of the invention is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and compounds of Applicants' invention. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

The compounds described herein can be used in combination with oneanother, with other active agents (e.g. anti-cancer agents) known to beuseful in treating a disease described herein (e.g. cancer), or withadjunctive agents that may not be effective alone, but may contribute tothe efficacy of the active agent.

In some embodiments, co-administration includes administering one activeagent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a secondactive agent (e.g. anti-cancer agent). Co-administration includesadministering two active agents simultaneously, approximatelysimultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes ofeach other), or sequentially in any order. In some embodiments,co-administration can be accomplished by co-formulation, i.e., preparinga single pharmaceutical composition including both active agents. Inother embodiments, the active agents can be formulated separately. Inanother embodiment, the active and/or adjunctive agents may be linked orconjugated to one another.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In some embodiments, ananti-cancer agent is a chemotherapeutic. In some embodiments, ananti-cancer agent is an agent identified herein having utility inmethods of treating cancer. In some embodiments, an anti-cancer agent isan agent approved by the FDA or similar regulatory agency of a countryother than the USA, for treating cancer. Examples of anti-cancer agentsinclude, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2)inhibitors (e.g. XL518, CI-1040, PD035901, selumetinib/AZD6244,GSK1120212/trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901,U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylatingagents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan,melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine,thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine, lomusitne, semustine, streptozocin), triazenes(decarbazine)), anti-metabolites (e.g., 5-azathioprine, leucovorin,capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folicacid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,fluorouracil, floxouridine, Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds or platinumcontaining agents (e.g. cisplatin, oxaloplatin, carboplatin),anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors ofmitogen-activated protein kinase signaling (e.g. U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies(e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, alltrans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), 5-aza-2′-deoxycytidine, all transretinoic acid, doxorubicin, vincristine, etoposide, gemcitabine,imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or rlL.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g. Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), Vincristine sulfate, Cryptophycin 52 (i.e. LY-355703),Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969),Oncocidin A1 (i.e. BTO-956 and DIME), Fijianolide B, Laulimalide,Narcosine (also known as NSC-5366), Nascapine, Hemiasterlin, Vanadoceneacetylacetonate, Monsatrol, Inanocine (i.e. NSC-698666), Eleutherobins(such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A,and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B,Diazonamide A, Taccalonolide A, Diozostatin, (−)-Phenylahistin (i.e.NSCL-96F037), Myoseverin B, Resverastatin phosphate sodium, steroids(e.g., dexamethasone), finasteride, aromatase inhibitors,gonadotropin-releasing hormone agonists (GnRH) such as goserelin orleuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate, megestrol acetate, medroxyprogesteroneacetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol),antiestrogen (e.g., tamoxifen), androgens (e.g., testosteronepropionate, fluoxymesterone), antiandrogen (e.g., flutamide),immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole,interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g.,anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonalantibodies), immunotoxins (e.g., anti-CD33 monoclonalantibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, hormonal therapies, or the like.

“Analog” and “analogue” are used interchangeably and are used inaccordance with their plain ordinary meaning within Chemistry andBiology and refers to a chemical compound that is structurally similarto another compound (i.e., a so-called “reference” compound) but differsin composition, e.g., in the replacement of one atom by an atom of adifferent element, or in the presence of a particular functional group,or the replacement of one functional group by another functional group,or the absolute stereochemistry of one or more chiral centers of thereference compound, including isomers thereof. Accordingly, an analog isa compound that is similar or comparable in function and appearance butnot in structure or origin to a reference compound.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments,about means within a standard deviation using measurements generallyacceptable in the art. In embodiments, about means a range extending to+/−10% of the specified value. In embodiments, about means the specifiedvalue.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature. Sulfur-containing amino acids refers tonaturally occurring and synthetic amino acids comprising sulfur, e.g.,methionine, cysteine, homocysteine, and taurine.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

Fanconi anemia is a disease characterized by developmentalabnormalities, bone marrow failure and cancer predisposition. Inembodiments, Fanconi anemia is referred to as Fanconi hypoplasticanemia, Fanconi pancytopenia, or Fanconi panmyelopathy.

The term “sensitize” or “sensitizing” as used herein in reference tocancer cells susceptibility to cancer therapies (e.g., chemotherapeuticagent, radiation, or a combination thereof) means positively affecting(e.g., increasing) the susceptibility of the cancer cells (e.g.,improving the efficacy of the cancer therapies) relative to thesusceptibility of the cancer cells in the absence of the compound asdescribed herein. In embodiments, sensitizing permits lower doses ofcancer therapies (e.g., chemotherapeutic agent, radiation, or acombination thereof) relative to the absence of sensitizing to betherapeutically effective.

In embodiments, a substituted or unsubstituted moiety (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, and/orsubstituted or unsubstituted heteroarylene) is unsubstituted (e.g., isan unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, unsubstituted alkylene, unsubstitutedheteroalkylene, unsubstituted cycloalkylene, unsubstitutedheterocycloalkylene, unsubstituted arylene, and/or unsubstitutedheteroarylene, respectively). In embodiments, a substituted orunsubstituted moiety (e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, and/or substituted or unsubstituted heteroarylene) issubstituted (e.g., is a substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl,substituted heteroaryl, substituted alkylene, substitutedheteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene, respectively).

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,wherein if the substituted moiety is substituted with a plurality ofsubstituent groups, each substituent group may optionally be different.In embodiments, if the substituted moiety is substituted with aplurality of substituent groups, each substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one size-limited substituentgroup, wherein if the substituted moiety is substituted with a pluralityof size-limited substituent groups, each size-limited substituent groupmay optionally be different. In embodiments, if the substituted moietyis substituted with a plurality of size-limited substituent groups, eachsize-limited substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one lower substituent group,wherein if the substituted moiety is substituted with a plurality oflower substituent groups, each lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of lower substituent groups, each lower substituent group isdifferent.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted moiety is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent group isdifferent.

Where a moiety is substituted (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene), the moiety is substituted with at least one substituent(e.g., a substituent group, a size-limited substituent group, or lowersubstituent group) and each substituent is optionally different.Additionally, where multiple substituents are present on a moiety, eachsubstituent may be optionally differently.

Compositions

Provided herein are compounds that are, inter alia, useful for thetreatment of cancer or Fanconi anemia.

In an aspect is provided a compound or pharmaceutically acceptable saltthereof, having the formula:

R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. The symbol n is an integer from1 to 4.

In embodiments, the disclosure provides compounds of formula (I) or apharmaceutically acceptable salt thereof, wherein the substituents areas defined herein.

In embodiments, the disclosure provides compounds of formula (II) or apharmaceutically acceptable salt thereof, wherein the substituents areas defined herein.

In embodiments, L is a bond, —C(O)—NH—, —NH—C(O)—, —NH—, —O—, —S—,—S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—, —CH₂N(C(O)OH)—, —CH₂—N(CH(O))—,—CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—, —CH₂NHC(O)—, —CH₂NHS(O)—,—CH₂NHS(O)₂—, substituted (e.g., substituted with substituent group(s),size-limited substituent group(s), or lower substituent group(s)) orunsubstituted alkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted heteroalkylene, substituted (e.g.,substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted cycloalkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheterocycloalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted arylene, or substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heteroarylene.

In embodiments, L is-C(O)—NH—, —NH—C(O)—, —C(O)—, —C(O)—O, —NH—, —O—,—S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—, —CH₂N(C(O)OH)—,—CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—, —CH₂NHC(O)—,—CH₂NHS(O)—, —CH₂NHS(O)₂—, unsubstituted C₁-C₆ alkylene, unsubstitutedC₁-C₆ heteroalkylene, unsubstituted 5-6 membered cycloalkylene, 5 to 6membered unsubstituted heterocycloalkylene, 5-6 membered unsubstitutedarylene, or 5 to 6 membered or unsubstituted heteroarylene. Inembodiments, L is —C(O)—NH—, —NH—C(O)—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—,—NH—S(O)—, —NH—S(O)₂—, —CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—,—CH₂NH—, —CH₂C(O)NH—, —CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—,unsubstituted C₁-C₆ alkylene, unsubstituted C₁-C₆ heteroalkylene,unsubstituted 5-6 membered cycloalkylene, 5 to 6 membered unsubstitutedheterocycloalkylene, 5-6 membered unsubstituted arylene, or 5 to 6membered or unsubstituted heteroarylene.

In embodiments, L is —C(O)—NH—, —C(O)—, —C(O)—O—, —NH—, —O—, —S—,—S(O)—, or —S(O)₂—. In embodiments, L is —C(O)—NH—, —NH—, —O—, —S—,—S(O)—, or —S(O)₂—. In embodiments, L is —C(O)—NH— or —C(O)—O—. Inembodiments, L is —C(O)—NH—.

In embodiments, R¹, is hydrogen, halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹,—S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰, —NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴,—NR¹⁵, —OCOR¹⁶, R¹⁷-substituted or unsubstituted alkyl (e.g. C₁-C₈alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), R¹⁷-substituted or unsubstitutedheteroalkyl (e.g. 2 to 8 membered heteroalkyl, 2 to 6 memberedheteroalkyl, or 2 to 4 membered heteroalkyl), R¹⁷-substituted orunsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈ cycloalkyl, orC₅-C₆ cycloalkyl), R¹⁷-substituted or unsubstituted heterocycloalkyl(e.g. 3 to 8 membered heterocycloalkyl, 4 to 8 memberedheterocycloalkyl, or 5 to 6 membered heterocycloalkyl), R¹⁷-substitutedor unsubstituted aryl (e.g. C₆-C₁₀ aryl or C₆ aryl), or R¹⁷-substitutedor unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl, 5 to 9membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R¹is hydrogen.

R¹⁷ is oxo, halogen, —F, —Cl, —Br, —I, —CF₃, —CCl₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl (e.g. C₁-C₈ alkyl, C1-C₆ alkyl, orC₁-C₄ alkyl), unsubstituted heteroalkyl (e.g. 2 to 8 memberedheteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 memberedheteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted heterocycloalkyl (e.g. 3to 8 membered heterocycloalkyl, 4 to 8 membered heterocycloalkyl, or 5to 6 membered heterocycloalkyl), unsubstituted aryl (e.g. C₆-C₁₀ aryl orC₆ aryl), or unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl,5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

In embodiments, R² is hydrogen, halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹,—S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰, —NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴,—NR¹⁵, —OCOR¹⁶, R¹⁸-substituted or unsubstituted alkyl (e.g. C₁-C₈alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), R¹⁸-substituted or unsubstitutedheteroalkyl (e.g. 2 to 8 membered heteroalkyl, 2 to 6 memberedheteroalkyl, or 2 to 4 membered heteroalkyl), R¹⁸-substituted orunsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈ cycloalkyl, orC₅-C₆ cycloalkyl), R¹⁸-substituted or unsubstituted heterocycloalkyl(e.g. 3 to 8 membered heterocycloalkyl, 4 to 8 memberedheterocycloalkyl, or 5 to 6 membered heterocycloalkyl), R¹⁸-substitutedor unsubstituted aryl (e.g. C₆-C₁₀ aryl or C₆ aryl), or R¹⁸-substitutedor unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl, 5 to 9membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R²is hydrogen.

R¹⁸ is oxo, halogen, —F, —Cl, —Br, —I, —CF₃, —CCl₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl (e.g. C₁-C₈ alkyl, C₁-C₆ alkyl, orC₁-C₄ alkyl), unsubstituted heteroalkyl (e.g. 2 to 8 memberedheteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 memberedheteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted heterocycloalkyl (e.g. 3to 8 membered heterocycloalkyl, 4 to 8 membered heterocycloalkyl, or 5to 6 membered heterocycloalkyl), unsubstituted aryl (e.g. C₆-C₁₀ aryl orC₆ aryl), or unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl,5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

In embodiments, R³, is hydrogen, halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹,—S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰, —NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴,—NR¹⁵, —OCOR¹⁶, R¹⁹-substituted or unsubstituted alkyl (e.g. C₁-C₈alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), R¹⁹-substituted or unsubstitutedheteroalkyl (e.g. 2 to 8 membered heteroalkyl, 2 to 6 memberedheteroalkyl, or 2 to 4 membered heteroalkyl), R¹⁹-substituted orunsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈ cycloalkyl, orC5-C6 cycloalkyl), R¹⁹-substituted or unsubstituted heterocycloalkyl(e.g. 3 to 8 membered heterocycloalkyl, 4 to 8 memberedheterocycloalkyl, or 5 to 6 membered heterocycloalkyl), R¹⁹-substitutedor unsubstituted aryl (e.g. C₆-C₁₀ aryl or C₆ aryl), or R¹⁹-substitutedor unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl, 5 to 9membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R³is hydrogen.

R¹⁹ is oxo, halogen, —F, —Cl, —Br, —I, —CF₃, —CCl₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl (e.g. C₁-C₈ alkyl, C₁-C₆ alkyl, orC₁-C₄ alkyl), unsubstituted heteroalkyl (e.g. 2 to 8 memberedheteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 memberedheteroalkyl), unsubstituted cycloalkyl (e.g. C₃-C₈ cycloalkyl, C₄-C₈cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted heterocycloalkyl (e.g. 3to 8 membered heterocycloalkyl, 4 to 8 membered heterocycloalkyl, or 5to 6 membered heterocycloalkyl), unsubstituted aryl (e.g. C₆-C₁₀ aryl orC₆ aryl), or unsubstituted heteroaryl (e.g. 5 to 10 membered heteroaryl,5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

In embodiments, R¹, R², and R³ are independently hydrogen, halogen,—NH₂, —OH, —NO₂, —C(O)CH₃, —NHC(O)CH₃, —OC(O)CH₃, or unsubstituted C₁-C₄alkyl. In embodiments, R¹, R², and R³ are hydrogen. In embodiments, R¹,R², and R³ are independently hydrogen, and L is —C(O)NH—.

In embodiments, X is hydrogen, halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹,—S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰, —NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴,—NR¹⁵, or —OCOR¹⁶. In embodiments, X is substituted or unsubstitutedalkylene (e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), substituted or unsubstituted cycloalkylene (e.g. C₃-C₈cycloalkylene, C₄-C₈ cycloalkylene, or C₅-C₆ cycloalkylene), substitutedor unsubstituted heterocycloalkylene (e.g. 3 to 8 memberedheterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5 to 6membered heterocycloalkylene), substituted or unsubstituted arylene(e.g. C₆-C₁₀ arylene or C₆ arylene), or substituted or unsubstitutedheteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9 memberedheteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, X is substituted C₁-C₈ alkyl, substituted 2-8 memberedheteroalkyl, substituted C₃-C₈ cycloalkyl, substituted 3-6 memberedheterocycloalkyl, substituted phenyl, or substituted 5 or 6 memberedheteroaryl. In embodiments, X is unsubstituted C₁-C₈ alkyl,unsubstituted 2-8 membered heteroalkyl, unsubstituted C₃-C₈ cycloalkyl,unsubstituted 3-6 membered heterocycloalkyl, unsubstituted phenyl, orunsubstituted 5 or 6 membered heteroaryl. In embodiments, X is asubstituted or unsubstituted C₁-C₈ alkyl. In embodiments, X is asubstituted or unsubstituted C₁-C₆ alkyl. In embodiments, X is asubstituted or unsubstituted C₁-C₄ alkyl. In embodiments, X is asubstituted or unsubstituted C₄ alkyl. In embodiments, X is anunsubstituted C₁-C₈ alkyl. In embodiments, X is an unsubstituted C₁-C₆alkyl. In embodiments, X is an unsubstituted C₁-C₄ alkyl. Inembodiments, X is an unsubstituted C₄ alkyl. In embodiments, X is —CH₃.In embodiments, X is a substituted C₁-C₈ alkyl. In embodiments, X is asubstituted C₁-C₆ alkyl. In embodiments, X is a substituted C₁-C₄ alkyl.In embodiments, X is a substituted C₄ alkyl.

In embodiments, X is R^(2a)-substituted or unsubstituted C₁-C₈ alkyl,R^(2a)-substituted or unsubstituted 2 to 8 membered heteroalkyl,R^(2a)-substituted or unsubstituted C₃-C₈ cycloalkyl, R^(2a)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(2a)-substituted orunsubstituted phenyl, or R^(2a)-substituted or unsubstituted 5 or 6membered heteroaryl. In embodiments, X is a R^(2a)-substituted orunsubstituted C₁-C₈ alkyl. In embodiments, X is a R^(2a)-substituted orunsubstituted C₁-C₆ alkyl. In embodiments, X is a R^(2a)-substituted orunsubstituted C₁-C₄ alkyl. In embodiments, X is a R^(2a)-substituted orunsubstituted C₄ alkyl. In embodiments, X is a R^(2a)-substituted C₁-C₈alkyl. In embodiments, X is a R^(2a)-substituted C₁-C₆ alkyl. Inembodiments, X is a R^(2a)-substituted C₁-C₄ alkyl. In embodiments, X isa R^(2a)-substituted C₄ alkyl. In embodiments, X is a R^(2a)-substitutedC₁ alkyl. In embodiments, X has the formula:

wherein R^(2a) is as described herein. In embodiments, X has theformula:

In embodiments, X has the formula:

R^(2a) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, R^(2b)-substitutedor unsubstituted C₁-C₈ alkyl, R^(2b-)substituted or unsubstituted 2-8membered heteroalkyl, R^(2b)-substituted or unsubstituted C₃-C₈cycloalkyl, R^(2b)-substituted or unsubstituted 3-6 memberedheterocycloalkyl, R^(2b)-substituted or unsubstituted phenyl, orR^(2b)-substituted or unsubstituted 5 or 6 membered heteroaryl. Inembodiments, R^(2a) is independently halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, R^(2b)-substitutedor unsubstituted C₁-C₈ alkyl, R^(2b)-substituted or unsubstituted 2-8membered heteroalkyl, R^(2b)-substituted or unsubstituted C₃-C₈cycloalkyl, R^(2b)-substituted or unsubstituted 3-6 memberedheterocycloalkyl, R^(2b)-substituted or unsubstituted phenyl, orR^(2b-)substituted or unsubstituted 5 or 6 membered heteroaryl.

In embodiments, R^(2a) is a R^(2b)-substituted or unsubstituted 3-6membered heterocycloalkyl, R^(2b)-substituted or unsubstituted phenyl,or R^(2b)-substituted or unsubstituted 5 or 6 membered heteroaryl. Inembodiments, R^(2a) is an unsubstituted 3-6 membered heterocycloalkyl,unsubstituted phenyl, or unsubstituted 5 or 6 membered heteroaryl. Inembodiments, R^(2a) is an unsubstituted 5 or 6 membered heteroaryl. Inembodiments, R^(2a) is an unsubstituted pyridinyl. In embodiments,R^(2a) is —C(CF₃)₂OH. In embodiments, R^(2a) is —C(CF₃)₃.

R^(2b) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, unsubstituted C₁-C₈alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 3-6 membered heterocycloalkyl, unsubstitutedphenyl, or unsubstituted 5 or 6 membered heteroaryl. In embodiments,R^(2b) is independently halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂. In embodiments, R^(2b) isindependently —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —CHO, —OH, —NH₂, —COOH, or—CONH₂. In embodiments, R^(2b) is independently —CF₃ or —OH.

In embodiments, R⁹ is independently hydrogen, halogen, —CN, —CF₃, —CCl₃,—CBr₃, —CI₃, —OH, —NH₂, R²⁰-substituted or unsubstituted alkylene (e.g.C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene), R²⁰-substituted orunsubstituted heteroalkylene (e.g. 2 to 10 membered heteroalkylene, 2 to8 membered heteroalkylene, 4 to 8 membered heteroalkylene, 2 to 6membered heteroalkylene, or 2 to 4 membered heteroalkylene),R²⁰-substituted or unsubstituted cycloalkylene (e.g. C₃-C₈cycloalkylene, C₄-C₈ cycloalkylene, or C₅-C₆ cycloalkylene),R²⁰-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²⁰-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²⁰-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹⁰ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²¹-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²¹-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²¹-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C₅-C₆ cycloalkylene),R²¹-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²¹-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²¹-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹¹ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²²-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²²-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²²-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C₅-C₆ cycloalkylene),R²²-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²²-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²²-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹¹ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²²-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²²-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²²-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C₅-C₆ cycloalkylene),R²²-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²²-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²²-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹² is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²³-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²³-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²³-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C5-C6 cycloalkylene),R²³-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²³-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²³-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹³ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²⁴-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²⁴-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²⁴-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C5-C6 cycloalkylene),R²⁴-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²⁴-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²⁴-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹⁴ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²⁵-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²⁵-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²⁵-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C5-C6 cycloalkylene),R²⁵-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²⁵-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²⁵-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹⁵ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²⁶-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²⁶-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²⁶-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C5-C6 cycloalkylene),R²⁶-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²⁶-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²⁶-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

In embodiments, R¹⁶ is independently hydrogen, halogen, —CN, —CF₃,—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, R²⁷-substituted or unsubstituted alkylene(e.g. C₁-C₈ alkylene, C₁-C₆ alkylene, or C₁-C₄ alkylene),R²⁷-substituted or unsubstituted heteroalkylene (e.g. 2 to 10 memberedheteroalkylene, 2 to 8 membered heteroalkylene, 4 to 8 memberedheteroalkylene, 2 to 6 membered heteroalkylene, or 2 to 4 memberedheteroalkylene), R²⁷-substituted or unsubstituted cycloalkylene (e.g.C₃-C₈ cycloalkylene, C₄-C₈ cycloalkylene, or C5-C6 cycloalkylene),R²⁷-substituted or unsubstituted heterocycloalkylene (e.g. 3 to 8membered heterocycloalkylene, 4 to 8 membered heterocycloalkylene, or 5to 6 membered heterocycloalkylene), R²⁷-substituted or unsubstitutedarylene (e.g. C₆-C₁₀ arylene or C₆ arylene), or R²⁷-substituted orunsubstituted heteroarylene (e.g. 5 to 10 membered heteroarylene, 5 to 9membered heteroarylene, or 5 to 6 membered heteroarylene).

R²⁰, R²¹, R²², R²³, R²⁴, R^(25′) R²⁶, and R²⁷ are independently oxo,halogen, —F, —Cl, —Br, —I, —CF₃, —CCl₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstitutedalkyl (e.g. C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstitutedheteroalkyl (e.g. 2 to 8 membered heteroalkyl, 2 to 6 memberedheteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl(e.g. C₃-C₈ cycloalkyl, C₄-C₈ cycloalkyl, or C₅-C₆ cycloalkyl),unsubstituted heterocycloalkyl (e.g. 3 to 8 membered heterocycloalkyl, 4to 8 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),unsubstituted aryl (e.g. C₆-C₁₀ aryl or C₆ aryl), or unsubstitutedheteroaryl (e.g. 5 to 10 membered heteroaryl, 5 to 9 memberedheteroaryl, or 5 to 6 membered heteroaryl).

In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3.In embodiments, n is 4.

In embodiments, the compound has the formula:

wherein R¹, R², R³, and X are as described herein.

In embodiments, the compound has the formula:

wherein X is as described herein.

In embodiments, the compound has the formula:

wherein X is a substituted or unsubstituted alkyl or a substituted orunsubstituted heteroalkyl.

In embodiments, the compound has the formula:

wherein Ring A is a substituted or unsubstituted cycloalkyl, substitutedor unsubstituted heterocycloalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl.

In embodiments, Ring A is R^(2a)-substituted or unsubstituted (C₆-C₁₀)aryl or R^(2a)-substituted or unsubstituted 5 to 10 membered heteroaryl.Ring A may be R^(2a)-substituted or unsubstituted (C₆-C₁₀) aryl. Ring Amay be R^(2a)-substituted or unsubstituted phenyl. Ring A may beR^(2a)-substituted or unsubstituted napthyl. Ring A may beR^(2a)-substituted or unsubstituted 5 to 10 membered heteroaryl. Ring Amay be R^(2a)-substituted or unsubstituted 5 to 6 membered heteroaryl.Ring A may be R^(2a)-substituted or unsubstituted thienyl. Ring A may beR^(2a)-substituted or unsubstituted furanyl. Ring A may beR^(2a)-substituted or unsubstituted pyrrolyl. Ring A may beR^(2a)-substituted or unsubstituted imidazolyl. Ring A may beR^(2a)-substituted or unsubstituted pyrazolyl. Ring A may beR^(2a)-substituted or unsubstituted oxazolyl. Ring A may beR^(2a)-substituted or unsubstituted isoxazolyl. Ring A may beR^(2a)-substituted or unsubstituted thiazolyl. Ring A may beR^(2a)-substituted or unsubstituted pyridinyl. Ring A may beR^(2a)-substituted or unsubstituted pyridyl. Ring A may beR^(2a)-substituted or unsubstituted pyrazinyl. Ring A may beR^(2a)-substituted or unsubstituted pyrimidinyl. Ring A may beR^(2a)-substituted or unsubstituted pyridazinyl. Ring A may beR^(2a)-substituted or unsubstituted 1,2,3-triazinyl. Ring A may beR^(2a)-substituted or unsubstituted 1,2,4-triazinyl. Ring A may beR^(2a)-substituted or unsubstituted 1,3,5-triazinyl. In embodiments,Ring A is R^(2a)-substituted (C₆-C₁₀) aryl or R^(2a)-substituted 5 to 10membered heteroaryl. Ring A may be R^(2a)-substituted (C₆-C₁₀) aryl.Ring A may be R^(2a)-substituted phenyl. Ring A may beR^(2a)-substituted napthyl. Ring A may be R^(2a)-substituted 5 to 10membered heteroaryl. Ring A may be R^(2a)-substituted 5 to 6 memberedheteroaryl. Ring A may be R^(2a)-substituted thienyl. Ring A may beR^(2a)-substituted furanyl. Ring A may be R^(2a)-substituted pyrrolyl.Ring A may be R^(2a)-substituted imidazolyl. Ring A may beR^(2a)-substituted pyrazolyl. Ring A may be R^(2a)-substituted oxazolyl.Ring A may be R^(2a)-substituted isoxazolyl. Ring A may beR^(2a)-substituted thiazolyl. Ring A may be R^(2a)-substitutedpyridinyl. Ring A may be R^(2a)-substituted pyridyl. Ring A may beR^(2a)-substituted pyrazinyl. Ring A may be R^(2a)-substitutedpyrimidinyl. Ring A may be R^(2a)-substituted pyridazinyl. Ring A may beR^(2a)-substituted 1,2,3-triazinyl. Ring A may be R^(2a)-substituted1,2,4-triazinyl. Ring A may be R^(2a)-substituted 1,3,5-triazinyl. RingA may be substituted with one R^(2a). Ring A may be substituted with twooptionally different R^(2a) substituents. Ring A may be substituted withthree optionally different R^(2a) substituents. Ring A may besubstituted with four optionally different R^(2a) substituents. Ring Amay be substituted with five optionally different R^(2a) substituents.

Ring A may be unsubstituted thienyl. Ring A may be unsubstitutedfuranyl. Ring A may be unsubstituted pyrrolyl. Ring A may beunsubstituted imidazolyl. Ring A may be unsubstituted pyrazolyl. Ring Amay be unsubstituted oxazolyl. Ring A may be unsubstituted isoxazolyl.Ring A may be unsubstituted thiazolyl. Ring A may be unsubstitutedpyridinyl. Ring A may be unsubstituted pyridyl. Ring A may beunsubstituted pyrazinyl. Ring A may be unsubstituted pyrimidinyl. Ring Amay be unsubstituted pyridazinyl. Ring A may be unsubstituted1,2,3-triazinyl. Ring A may be unsubstituted 1,2,4-triazinyl. Ring A maybe unsubstituted 1,3,5-triazinyl.

In embodiments, the compound has the formula:

wherein R^(2a) is as defined herein and the symbol z1 is an integer from0 to 5. In embodiments, z1 is 0 or 1. In embodiments, z1 is 0. Inembodiments, z1 is 1. In embodiments, z1 is 2. In embodiments, z1 is 3.In embodiments, z1 is 4. In embodiments, z1 is 5.

In embodiments, the compound has the formula:

In embodiments, the compound has the formula:

In embodiments, the compound has the formula:

(also referred to herein as C5-1).

In embodiments, the compound has the formula:

(also referred to herein as C5-2).

In embodiments, the compound has the formula:

(also referred to herein as C5-3).

In embodiments, there is a proviso that the compound of Formula (I) isnot a compound wherein the substituents are concurrently the following:R¹, R², and R³ are hydrogen; L is —C(O)—; X is —OR¹⁴ and R¹⁴ ishydrogen. In embodiments, the compound is not:

In embodiments, the compound is not:

In embodiments, the compound is not:

Pharmaceutical Compositions

In another aspect, is provided a pharmaceutical composition including apharmaceutically acceptable excipient and a compound as described hereinor a pharmaceutically acceptable salt thereof.

The pharmaceutical compositions may include optical isomers,diastereomers, or pharmaceutically acceptable salts of the compoundsdisclosed herein. The compound included in the pharmaceuticalcomposition may be covalently attached to a carrier moiety.Alternatively, the compound included in the pharmaceutical compositionis not covalently linked to a carrier moiety.

The compounds of the present invention can be prepared and administeredin a wide variety of oral, parenteral and topical dosage forms. Oralpreparations include tablets, pills, powder, dragees, capsules, liquids,lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. The compounds of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present invention can be administered transdermally. Itis also envisioned that multiple routes of administration (e.g.,intramuscular, oral, transdermal) can be used to administer thecompounds of the invention. Accordingly, the present invention alsoprovides pharmaceutical compositions comprising a pharmaceuticallyacceptable excipient and one or more compounds of the invention.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. When administered in methods to treat a disease, suchcompositions will contain an amount of active ingredient effective toachieve the desired result, e.g., modulating the activity of a targetmolecule, and/or reducing, eliminating, or slowing the progression ofcancer symptoms. Determination of a therapeutically effective amount ofa compound of the invention is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureherein.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated (e.g., cancer, breast cancer, colon cancer, prostatecancer, lung cancer, or ovarian cancer), kind of concurrent treatment,complications from the disease being treated or other health-relatedproblems. Other therapeutic regimens or agents can be used inconjunction with the methods and compounds described herein. Adjustmentand manipulation of established dosages (e.g., frequency and duration)are well within the ability of those skilled in the art.

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

Certain compositions described herein of the present invention can existin unsolvated forms as well as solvated forms, including hydrated forms.In general, the solvated forms are equivalent to unsolvated forms andare intended to be encompassed within the scope of the presentinvention. Certain compounds of the present invention may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated by the present invention andare intended to be within the scope of the present invention.

In another embodiment, the compositions of the present invention areuseful for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly include asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The compounds described herein can be used in combination with oneanother, with other active agents known to be useful in treating adisease associated with cells expressing folate transporters (e.g.breast cancer, colon cancer, prostate cancer, lung cancer, or ovariancancer), or with adjunctive agents that may not be effective alone, butmay contribute to the efficacy of the active agent.

In embodiments, the compositions and compounds of Formula (I) describedherein can be co-administered with conventional chemotherapeutic agentsincluding alkylating agents (e.g., cyclophosphamide, ifosfamide,chlorambucil, busulfan, melphalan, mechlorethamine, uramustine,thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5-fluorouracil,azathioprine, methotrexate, leucovorin, capecitabine, cytarabine,floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.),plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine,podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors(e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin), and the like. In embodiments, thecompound of Formula (I) is administered prior to the chemotherapeuticagent.

In other embodiments, the pharmaceutical compositions described hereincomprise a compound of Formula (I) or a pharmaceutically acceptable saltthereof, a chemotherapeutic agent (such as alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.),anti-metabolites (e.g., 5-fluorouracil, azathioprine, methotrexate,leucovorin, capecitabine, cytarabine, floxuridine, fludarabine,gemcitabine, pemetrexed, raltitrexed, etc.), plant alkaloids (e.g.,vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin,paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g.,irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate,teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin,daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g.cisplatin, oxaloplatin, carboplatin); and a pharmaceutically acceptableexcipient.

The compounds or drugs described herein can also be co-administered withconventional hormonal therapeutic agents including, but not limited to,steroids (e.g., dexamethasone), finasteride, aromatase inhibitors,tamoxifen, and gonadotropin-releasing hormone agonists (GnRH) such asgoserelin. In embodiments, the compound of Formula (I) is administeredprior to the hormonal therapeutic agent.

In other embodiments, the pharmaceutical compositions described hereincomprise a compound of Formula (I) or (II) or a pharmaceuticallyacceptable salt thereof; a hormonal therapeutic agent (including, butnot limited to, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and gonadotropin-releasing hormone agonists(GnRH) such as goserelin); and a pharmaceutically acceptable excipient.

The compounds or drugs described herein can also be co-administered withPARP inhibiting agents (e.g., PARP inhibitors) including, but notlimited to 4-iodo-3-nitrobenzamide,4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one,8-Fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one,2-((R)-2-Methylpyrrolidin-2-yl)-1H-benzimidazole-4-carboxamide, and3-aminobenzamide. In embodiments, the compound of Formula (I) or (II) isadministered prior to the PARP inhibiting agent.

In other embodiments, the pharmaceutical compositions described hereincomprise a compound of Formula (I) or (II) or a pharmaceuticallyacceptable salt thereof; a PARP inhibiting agent (including, but notlimited to 4-iodo-3-nitrobenzamide,4-[(3-[(4-cyclopropylcarbonyl)piperazin-4-yl]carbonyl)-4-fluorophenyl]methyl(2H)phthalazin-1-one,8-Fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one,2-((R)-2-Methylpyrrolidin-2-yl)-1H-benzimidazole-4-carboxamide, and3-aminobenzamide); and a pharmaceutically acceptable excipient.

In a further embodiment, the compounds or drugs described herein can beco-administered with conventional radiopharmaceuticals including, butnot limited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y,⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens. In embodiments, the compound of Formula (I) or(II) is administered prior to the radiopharmaceutical.

In other embodiments, the pharmaceutical compositions described hereincomprise a compound of Formula (I) or (II) or a pharmaceuticallyacceptable salt thereof; a radiopharmaceutical (including, but notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens); and a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques or maybe produced under sterile conditions. Aqueous solutions can be packagedfor use or filtered under aseptic +conditions and lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration. The compositions can contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, and the like, e.g., sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate, and triethanolamine oleate.

Formulations suitable for oral administration can comprise: (a) liquidsolutions, such as an effective amount of a packaged compound or drugsuspended in diluents, e.g., water, saline, or PEG 400; (b) capsules,sachets, or tablets, each containing a predetermined amount of acompound or drug, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, microcrystallinecellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,stearic acid, and other excipients, colorants, fillers, binders,diluents, buffering agents, moistening agents, preservatives, flavoringagents, dyes, disintegrating agents, and pharmaceutically compatiblecarriers. Lozenge forms can comprise a compound or drug in a flavor,e.g., sucrose, as well as pastilles comprising the polypeptide orpeptide fragment in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like, containing, inaddition to the polypeptide or peptide, carriers known in the art.

The compound or drug of choice, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which comprises an effective amount of a packagedcompound or drug with a suppository base. Suitable suppository basesinclude natural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichcontain a combination of the compound or drug of choice with a base,including, for example, liquid triglycerides, polyethylene glycols, andparaffin hydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Injection solutions and suspensions can also beprepared from sterile powders, granules, and tablets. In the practice ofthe present invention, compositions can be administered, for example, byintravenous infusion, orally, topically, intraperitoneally,intravesically, or intrathecally. Parenteral administration, oraladministration, and intravenous administration are the preferred methodsof administration. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component, e.g., a compound ordrug. The unit dosage form can be a packaged preparation, the packagecontaining discrete quantities of preparation, such as packeted tablets,capsules, and powders in vials or ampoules. Also, the unit dosage formcan be a capsule, tablet, cachet, or lozenge itself, or it can be theappropriate number of any of these in packaged form. The compositioncan, if desired, also contain other compatible therapeutic agents.

Methods of Treatment

In an aspect is provided, a method of treating cancer in a patient inneed thereof including administering to the patient a therapeuticallyeffective amount of a pharmaceutical composition as described herein totreat the cancer in the patient.

In an aspect is provided, a method of treating cancer in a patient inneed thereof including administering to the patient a therapeuticallyeffective amount of a compound as described herein, and achemotherapeutic agent, radiation, or a combination thereof, to treatthe cancer in the patient. In embodiments, the compound as describedherein is administered prior to the chemotherapeutic agent, radiation,or a combination thereof. In embodiments, the compound as describedherein is administered concurrently with the chemotherapeutic agent,radiation, or a combination thereof. The concurrent administration maybe separate pharmaceutical compositions or may be a singlepharmaceutical composition. The single pharmaceutical composition fortreating cancer may comprise a compound described herein or apharmaceutically acceptable salt thereof; one or more chemotherapeuticagents; and a pharmaceutically acceptable excipient. The singlepharmaceutical composition for treating cancer may comprise a compounddescribed herein or a pharmaceutically acceptable salt thereof; one ormore radiopharmaceuticals; and a pharmaceutically acceptable excipient.The single pharmaceutical composition for treating cancer may comprise acompound described herein or a pharmaceutically acceptable salt thereof;one or more chemotherapeutic agents; one or more radiopharmaceuticals;and a pharmaceutically acceptable excipient. The cancer may be breast,colon, prostate, lung, or ovarian cancer.

In an aspect is provided a method of sensitizing cancer cells toradiation therapy or chemotherapy including administering the compoundas described herein to the cancer cells in vitro or in vivo to sensitizethe cancer cells to radiation therapy or chemotherapy. In embodiments,the method of sensitizing cancer cells to radiation therapy orchemotherapy includes administering the compound as described herein tothe cancer cells in vitro to sensitize the cancer cells to radiationtherapy or chemotherapy. In embodiments, the method of sensitizingcancer cells to radiation therapy or chemotherapy includes administeringthe compound as described herein to the cancer cells in vivo tosensitize the cancer cells to radiation therapy or chemotherapy. Inembodiments, the method of sensitizing cancer cells to radiation therapyincludes administering the compound as described herein to the cancercells in vitro or in vivo to sensitize the cancer cells to radiationtherapy. In embodiments, the method of sensitizing cancer cells tochemotherapy includes administering the compound as described herein tothe cancer cells in vitro or in vivo to sensitize the cancer cells tochemotherapy.

In another aspect is provided a method of potentiating the clinicalefficacy of a PARP inhibitor including co-administering the compound asdescribed herein with the PARP inhibitor to potentiate the clinicalefficacy of the PARP inhibitor. In this embodiment, the PARP inhibitorwill exhibit improved clinical efficacy when co-administered with thecompounds described herein than if the PARP inhibitor was administeredwithout the compounds described herein.

Provided in an aspect is a method of potentiating the clinical efficacyof a topoisomerase inhibitor including administering the compound asdescribed herein in conjunction with the topoisomerase inhibitor topotentiate the clinical efficacy of the topoisomerase inhibitor. Inembodiments, the topoisomerase inhibitor is irinotecan, topotecan,camptothecin, lamellarin D, etoposide, teniposide, doxorubicin,daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylicacid, a quinolone synthesized from cannabidiol (e.g., HU-331), orplant-derived phenols (e.g., epigallocatechin gallate, genistein,quercetin, or resveratrol). In embodiments, the topoisomerase inhibitoris a topoisomerase I inhibitor. In embodiments, the topoisomeraseinhibitor is irinotecan, topotecan, camptothecin, lamellarin D. Inembodiments, the topoisomerase inhibitor is camptothecin. In thisembodiment, the topoisomerase inhibitor will exhibit improved clinicalefficacy when co-administered with the compounds described herein thanif the topoisomerase inhibitor was administered without the compoundsdescribed herein.

In embodiments, the chemotherapeutic agent is a PARP inhibitor,topoisomerase inhibitor, or topoisomerase I inhibitor. In embodiments,the chemotherapeutic agent is a PARP inhibitor. In embodiments, thechemotherapeutic agent is a topoisomerase inhibitor. In embodiments, thechemotherapeutic agent is a topoisomerase I inhibitor. In embodiments,the chemotherapeutic agent is irinotecan, topotecan, camptothecin,lamellarin D, etoposide, teniposide, doxorubicin, daunorubicin,mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, aquinolone synthesized from cannabidiol (e.g., HU-331), or plant-derivedphenols (e.g., epigallocatechin gallate, genistein, quercetin, orresveratrol).

In embodiments, the cancer is breast cancer, colon cancer, prostatecancer, lung cancer, or ovarian cancer. In embodiments, the cancer isbreast cancer. In embodiments, the cancer is colon cancer. Inembodiments, the cancer is prostate cancer. In embodiments, the canceris lung cancer. In embodiments, the cancer is ovarian cancer.

In an aspect is provided a method of inhibiting DNA replication in cellsincluding administering the compound as described herein in vitro or invivo to the cells to inhibit DNA replication. In embodiments, the methodof inhibiting DNA replication in cells includes administering thecompound as described herein in vitro to the cells to inhibit DNAreplication. In embodiments, the method of inhibiting DNA replication incells includes administering the compound as described herein in vivo tothe cells to inhibit DNA replication. In embodiments, the methodincludes interfering with telomere replication or repait.

In an aspect is provided a method of inhibiting DNA replication in apatient in need thereof including administering the compound asdescribed herein to the patient to inhibit DNA replication in thepatient.

Provided in an aspect is a method of suppressing DNA double-strand breakrepair end resection, recombination, over-resection of nascent DNA incells defective in fork protection, and restart of stalled DNAreplication forks in cells or a patient in need thereof includingadministering the compound as described herein in vitro or in vivo tocells or the patient to suppress DNA double-strand break repair endresection, recombination, over-resection of nascent DNA in cellsdefective in fork protection, and restart of stalled DNA replicationforks. In embodiments, the method of suppressing DNA double-strand breakrepair end resection, recombination, over-resection of nascent DNA incells defective in fork protection, and restart of stalled DNAreplication forks in cells or a patient in need thereof further includesadministering a chemotherapeutic agent, radiation, or a combinationthereof. In embodiments, the method of suppressing DNA double-strandbreak repair includes inhibiting replication, inhibiting resection,inhibiting telomere homeostasis, or inhibiting DNA protein activity. Inembodiments, the method includes interfering with telomere replicationor repair.

In an aspect is provided a pharmaceutical composition including thecompound as described herein; a chemotherapeutic agent; and apharmaceutically acceptable carrier.

In an aspect is provided, a pharmaceutical composition including thecompound as described herein; a PARP inhibitor; and a pharmaceuticallyacceptable carrier.

In an aspect is provided, a pharmaceutical composition including thecompound as described herein; a topoisomerase inhibitor; and apharmaceutically acceptable carrier.

In an aspect is provided, a pharmaceutical composition including thecompound as described herein; a radiopharmaceutical; and apharmaceutically acceptable carrier.

In an aspect is provided, a pharmaceutical composition including thecompound as described herein; a chemotherapeutic agent; aradiopharmaceutical; and a pharmaceutically acceptable carrier.

In an aspect is provided, a method of treating Fanconi anemia in apatient in need thereof including administering to the patient atherapeutically effective amount of the compound or pharmaceuticalcomposition as described herein. In embodiments, the patient further hascancer (e.g., leukemias and solid tumors of the head and neck). Inembodiments, the patient has both cancer (e.g., leukemias and solidtumors of the head and neck) and Fanconi anemia.

In embodiments of the methods described herein, the compound has theformula:

or a tautomer of any one of the foregoing.

In embodiments of the methods described herein, the compound has theformula:

or a tautomer thereof.

In embodiments of the methods described herein, the compound has theformula:

or a tautomer thereof.

In embodiments of the methods described herein, the compound has theformula:

or a tautomer thereof.

In embodiments of the methods described herein, the compound has theformula:

or a tautomer thereof.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EMBODIMENTS Embodiment P1

A compound of Formula (I):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment P2

The compound of Embodiment P1, wherein L is-C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, unsubstituted C₁-C₆ alkylene,unsubstituted C₁-C₆ heteroalkylene, unsubstituted 5-6 memberedcycloalkylene, 5-6 membered unsubstituted heterocycloalkylene, 5-6membered unsubstituted arylene, or 5-6 membered or unsubstitutedheteroarylene.

Embodiment P3

The compound of Embodiment P1, wherein L is —C(O)—NH—, —C(O)—, —C(O)—O—,—NH—, —O—, —S—, —S(O)—, or —S(O)₂—.

Embodiment P4

The compound of Embodiment P1, wherein L is —C(O)—NH— or —C(O)—O—.

Embodiment P5

The compound of Embodiment P1, wherein R¹, R², and R³ are eachindependently hydrogen, halogen, —NH₂, —OH, —NO₂, —C(O)CH₃, —NHC(O)CH₃,—OC(O)CH₃, or unsubstituted C₁₋₄ alkyl.

Embodiment P6

The compound of Embodiment P1, wherein R¹, R², and R³ are hydrogen.

Embodiment P7

The compound of Embodiment P1, wherein R¹, R², and R³ are hydrogen, andL is —C(O)NH—.

Embodiment P8

The compound of any one of Embodiments P1 to P7, wherein X is hydrogen,halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰,—C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, or —OCOR¹⁶.

Embodiment P9

The compound of any one of Embodiments P1 to P7, wherein X issubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

Embodiment P10

The compound of any one of Embodiments P1 to P7, wherein X isR^(2a)-substituted or unsubstituted C₁-C₈ alkyl, R^(2a)-substituted orunsubstituted 2-8 membered heteroalkyl, R^(2a)-substituted orunsubstituted C₃-C₈ cycloalkyl, R^(2a)-substituted or unsubstituted 3-6membered heterocycloalkyl, R^(2a)-substituted or unsubstituted phenyl,or R^(2a)-substituted or unsubstituted 5 or 6 membered heteroaryl;R^(2a) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, R^(2b)-substitutedor unsubstituted C₁-C₈ alkyl, R^(2b)-substituted or unsubstituted 2-8membered heteroalkyl, R^(2b)-substituted or unsubstituted C₃-C₈cycloalkyl, R^(2b)-substituted or unsubstituted 3-6 memberedheterocycloalkyl, R^(2b)-substituted or unsubstituted phenyl, orR^(2b)-substituted or unsubstituted 5 or 6 membered heteroaryl; andR^(2b) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, unsubstituted C₁-C₈alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 3-6 membered heterocycloalkyl, unsubstitutedphenyl, or unsubstituted 5 or 6 membered heteroaryl.

Embodiment P11

The compound of any one of Embodiments P1 to P7, wherein X isunsubstituted C₁-C₈ alkyl, unsubstituted 2-8 membered heteroalkyl,unsubstituted C₃-C₈ cycloalkyl, unsubstituted 3-6 memberedheterocycloalkyl, unsubstituted phenyl, or unsubstituted 5 or 6 memberedheteroaryl.

Embodiment P12

The compound of any one of Embodiments P1 to P7, wherein X issubstituted C₁-C₈ alkyl, substituted 2-8 membered heteroalkyl,substituted C₃-C₈ cycloalkyl, substituted 3-6 membered heterocycloalkyl,substituted phenyl, or substituted 5 or 6 membered heteroaryl.

Embodiment P13

The compound of Embodiment P1, with the proviso that the compound doesnot concurrently have substituents wherein R₁, R₂, and R₃ are hydrogen;L is —C(O)—; X is —OR₁₄; and R¹⁴ is hydrogen.

Embodiment P14

A compound of the formula:

Embodiment P15

A compound of the formula:

Embodiment P16

A compound of the formula:

Embodiment P17

A compound of the formula:

Embodiment P18

A pharmaceutical composition comprising the compound of any one ofEmbodiments P1 to P17 and a pharmaceutically acceptable carrier.

Embodiment P19

A method of treating cancer in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of acompound of any one of Embodiments P1 to P17 to treat the cancer in thepatient.

Embodiment P20

The method of Embodiment P19, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment P21

A method of treating cancer in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of apharmaceutical composition of Embodiment P18 to treat the cancer in thepatient.

Embodiment P22

The method of Embodiment P21, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment P23

A method of treating cancer in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of (i) acompound of any one of Embodiments P1 to P17, and (ii) achemotherapeutic agent, radiation, or a combination thereof, to treatthe cancer in the patient.

Embodiment P24

The method of Embodiment P23, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment P25

The method of Embodiment P23, wherein the chemotherapeutic agent is aPARP inhibitor.

Embodiment P26

The method of Embodiment P23, wherein the chemotherapeutic agent is atopoisomerase inhibitor.

Embodiment P27

The method of Embodiment P26, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.

Embodiment P28

The method of Embodiment P23, wherein (i) is administered prior to (ii).

Embodiment P29

The method of Embodiment P23, wherein (i) and (ii) are administeredconcurrently.

Embodiment P30

A method of sensitizing cancer cells to radiation therapy orchemotherapy comprising administering the compound of any one ofEmbodiments P1 to P17 to the cancer cells in vitro or in vivo tosensitize the cancer cells to radiation therapy or chemotherapy.

Embodiment P31

A method of potentiating the clinical efficacy of a PARP inhibitorcomprising administering the compound of any one of Embodiments P1 toP17 in conjunction with the PARP inhibitor to potentiate the clinicalefficacy of the PARP inhibitor.

Embodiment P32

A method of potentiating the clinical efficacy of a topoisomeraseinhibitor comprising administering the compound of any one ofEmbodiments P1 to P17 in conjunction with the topoisomerase inhibitor topotentiate the clinical efficacy of the topoisomerase inhibitor.

Embodiment P33

The method of Embodiment P32, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.

Embodiment P34

A method of inhibiting DNA replication in cells comprising administeringthe compound of any one of Embodiments P1 to P17 in vitro or in vivo tothe cells to inhibit DNA replication.

Embodiment P35

A method of inhibiting DNA replication in a patient in need thereofcomprising administering the compound of any one of Embodiments P1 toP17 to the patient to inhibit DNA replication in the patient.

Embodiment P36

A method of suppressing DNA double-strand break repair end resection,recombination, over-resection of nascent DNA in cells defective in forkprotection, and restart of stalled NDA replication forks in cells or apatient in need thereof comprising administering the compound of any oneof Embodiments P1 to P17 in vitro or in vivo to cells or the patient tosuppress DNA double-strand break repair end resection, recombination,over-resection of nascent DNA in cells defective in fork protection, andrestart of stalled NDA replication forks.

Embodiment P37

The method of Embodiment P36, further comprising administering achemotherapeutic agent, radiation, or a combination thereof.

Embodiment P38

A pharmaceutical composition comprising the compound of any one ofEmbodiments P1 to P17; a chemotherapeutic agent; and a pharmaceuticallyacceptable carrier.

Embodiment P39

A pharmaceutical composition comprising the compound of any one ofEmbodiments P1 to P17; a PARP inhibitor; and a pharmaceuticallyacceptable carrier.

Embodiment P40

A pharmaceutical composition comprising the compound of any one ofEmbodiments P1 to P17; a topoisomerase inhibitor; and a pharmaceuticallyacceptable carrier.

Embodiment P41

A method of treating Fanconi anemia in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of the compound of any one of Embodiments P1-P17.

Embodiment P42

A method of treating Fanconi anemia in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of the composition of Embodiment P18.

ADDITIONAL EMBODIMENTS Embodiment 1

A compound or a pharmaceutically acceptable salt thereof, wherein thecompound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4; wherein thecompound of formula (I) is not:

and wherein the compound of formula (II) is not:

Embodiment 2

The compound of Embodiment 1, wherein L is-C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, unsubstituted C₁-C₆ alkylene,unsubstituted C₁-C₆ heteroalkylene, unsubstituted 5-6 memberedcycloalkylene, 5-6 membered unsubstituted heterocycloalkylene, 5-6membered unsubstituted arylene, or 5-6 membered or unsubstitutedheteroarylene.

Embodiment 3

The compound of Embodiment 1, wherein L is-C(O)—NH—, —NH—C(O)—, —NH—,—O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—, —CH₂N(C(O)OH)—,—CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—, —CH₂NHC(O)—,—CH₂NHS(O)—, —CH₂NHS(O)₂—, unsubstituted C₁-C₆ alkylene, unsubstitutedC₁-C₆ heteroalkylene, unsubstituted 5-6 membered cycloalkylene, 5-6membered unsubstituted heterocycloalkylene, 5-6 membered unsubstitutedarylene, or 5-6 membered or unsubstituted heteroarylene.

Embodiment 4

The compound of Embodiment 1, wherein L is —C(O)—NH—, —C(O)—, —C(O)—O—,—NH—, —O—, —S—, —S(O)—, or —S(O)₂—. The compound of Embodiment 1,wherein L is —C(O)—NH—, —NH—, —O—, —S—, —S(O)—, or —S(O)₂—.

Embodiment 5

The compound of Embodiment 1, wherein L is —C(O)—NH— or —C(O)—O—.

Embodiment 6

The compound of Embodiment 1, wherein L is —C(O)—NH—.

Embodiment 7

The compound of Embodiment 1, wherein R¹, R², and R³ are eachindependently hydrogen, halogen, —NH₂, —OH, —NO₂, —C(O)CH₃, —NHC(O)CH₃,—OC(O)CH₃, or unsubstituted C₁₋₄ alkyl.

Embodiment 8

The compound of Embodiment 1, wherein R¹, R², and R³ are hydrogen.

Embodiment 9

The compound of Embodiment 1, wherein R¹, R², and R³ are hydrogen, and Lis —C(O)NH—.

Embodiment 10

The compound of any one of Embodiments 1 to 7, wherein X is hydrogen,halogen, —N₃, —NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰,—C(O)R¹¹, —C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, or —OCOR¹⁶.

Embodiment 11

The compound of any one of Embodiments 1 to 7, wherein X is substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

Embodiment 12

The compound of any one of Embodiments 1 to 7, wherein X is R^(2a)substituted or unsubstituted C₁-C₈ alkyl, R^(2a)-substituted orunsubstituted 2-8 membered heteroalkyl, R^(2a)-substituted orunsubstituted C₃-C₈ cycloalkyl, R^(2a)-substituted or unsubstituted 3-6membered heterocycloalkyl, R^(2a)-substituted or unsubstituted phenyl,or R^(2a)-substituted or unsubstituted 5 or 6 membered heteroaryl;R^(2a) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, R^(2b)-substitutedor unsubstituted C₁-C₈ alkyl, R^(2b)-substituted or unsubstituted 2-8membered heteroalkyl, R^(2b) substituted or unsubstituted C₃-C₈cycloalkyl, R^(2b)-substituted or unsubstituted 3-6 memberedheterocycloalkyl, R^(2b)-substituted or unsubstituted phenyl, orR^(2b)-substituted or unsubstituted 5 or 6 membered heteroaryl; andR^(2b) is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —CHO, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, unsubstituted C₁-C₈alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 3-6 membered heterocycloalkyl, unsubstitutedphenyl, or unsubstituted 5 or 6 membered heteroaryl.

Embodiment 13

The compound of any one of Embodiments 1 to 7, wherein X isunsubstituted C₁-C₈ alkyl, unsubstituted 2-8 membered heteroalkyl,unsubstituted C₃-C₈ cycloalkyl, unsubstituted 3-6 memberedheterocycloalkyl, unsubstituted phenyl, or unsubstituted 5 or 6 memberedheteroaryl.

Embodiment 14

The compound of any one of Embodiments 1 to 7, wherein X is substitutedC₁-C₈ alkyl, substituted 2-8 membered heteroalkyl, substituted C₃-C₈cycloalkyl, substituted 3-6 membered heterocycloalkyl, substitutedphenyl, or substituted 5 or 6 membered heteroaryl.

Embodiment 15

The compound of embodiment 1, having the formula:

Embodiment 16

The compound of embodiment 1, having the formula:

Embodiment 17

The compound of embodiment 1, having the formula:

Embodiment 18

A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound or a pharmaceutically acceptable salt thereof,wherein the compound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 19

A method of treating cancer in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of acompound or a pharmaceutically acceptable salt thereof, wherein thecompound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 20

The method of Embodiment 19, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment 21

A method of treating cancer in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of apharmaceutical composition of Embodiment 20 to treat the cancer in thepatient.

Embodiment 22

The method of Embodiment 23, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment 23

A method of treating cancer in a patient in need thereof comprising: (i)administering to the patient a therapeutically effective amount of acompound or a pharmaceutically acceptable salt thereof, wherein thecompound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4, and (ii) achemotherapeutic agent, radiation, or a combination thereof, to treatthe cancer in the patient.

Embodiment 24

The method of Embodiment 25, wherein the cancer is breast cancer, coloncancer, prostate cancer, lung cancer, or ovarian cancer.

Embodiment 25

The method of Embodiment 25, wherein the chemotherapeutic agent is aPARP inhibitor.

Embodiment 26

The method of Embodiment 25, wherein the chemotherapeutic agent is atopoisomerase inhibitor.

Embodiment 27

The method of Embodiment 28, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.

Embodiment 28

The method of Embodiment 25, wherein (i) is administered prior to (ii).

Embodiment 29

The method of Embodiment 25, wherein (i) and (ii) are administeredconcurrently.

Embodiment 30

A method of sensitizing cancer cells to radiation therapy orchemotherapy comprising administering a compound or a pharmaceuticallyacceptable salt thereof, wherein the compound has formula (I) or formula(II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 31

A method of potentiating the clinical efficacy of a PARP inhibitorcomprising administering a compound or a pharmaceutically acceptablesalt thereof, wherein the compound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 32

A method of potentiating the clinical efficacy of a topoisomeraseinhibitor comprising administering a compound or a pharmaceuticallyacceptable salt thereof, wherein the compound has formula (I) or formula(II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 33

The method of Embodiment 34, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.

Embodiment 34

A method of inhibiting DNA replication in cells comprising administeringa compound or a pharmaceutically acceptable salt thereof, wherein thecompound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 35

A method of inhibiting DNA replication in a patient in need thereofcomprising administering a compound or a pharmaceutically acceptablesalt thereof, wherein the compound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 36

A method of suppressing DNA double-strand break repair end resection,recombination, over-resection of nascent DNA in cells defective in forkprotection, and restart of stalled DNA replication forks in cells or apatient in need thereof comprising administering a compound or apharmaceutically acceptable salt thereof, wherein the compound hasformula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 37

The method of Embodiment 38, further comprising administering achemotherapeutic agent, radiation, or a combination thereof.

Embodiment 38

A pharmaceutical composition comprising a chemotherapeutic agent; apharmaceutically acceptable carrier; and a compound or apharmaceutically acceptable salt thereof, wherein the compound hasformula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 39

A pharmaceutical composition comprising a PARP inhibitor; apharmaceutically acceptable carrier; and a compound or apharmaceutically acceptable salt thereof, wherein the compound hasformula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 40

A pharmaceutical composition comprising a topoisomerase inhibitor; apharmaceutically acceptable carrier; and a compound or apharmaceutically acceptable salt thereof, wherein the compound hasformula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 41

A method of treating Fanconi anemia in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a compound or a pharmaceutically acceptable salt thereof,wherein the compound has formula (I) or formula (II):

wherein R¹, R², and R³ are each independently hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R″, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; L is a bond, —C(O)—NH—, —NH—C(O)—, —C(O)—,—C(O)—O—, —NH—, —O—, —S—, —S(O)—, —S(O)₂—, —NH—S(O)—, —NH—S(O)₂—,—CH₂N(C(O)OH)—, —CH₂—N(CH(O))—, —CH₂—N(SO₂)—, —CH₂NH—, —CH₂C(O)NH—,—CH₂NHC(O)—, —CH₂NHS(O)—, —CH₂NHS(O)₂—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; X is hydrogen, halogen, —N₃,—NHC═(O)NHNH₂, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —NR⁹R¹⁰, —C(O)R¹¹,—C(O)NR⁹R¹⁰, —NR¹²C(O)R¹¹, —S(O)_(n)R¹³, —S(O)_(n)NR⁹R¹⁰,—NR¹²S(O)_(n)R¹³, —NO₂, —OR¹⁴, —SR¹⁴, —NR¹⁵, —OCOR¹⁶, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹, R¹⁰R¹¹, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶, areindependently hydrogen, halogen, —CN, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and n is 1 to 4.

Embodiment 42

A method of treating Fanconi anemia in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of the composition of Embodiment 20.

Embodiment 43

The method of embodiment 41 or 42, wherein the patient has cancer.

EXAMPLES

DNA replication is the central process of all actively dividing cells.Blocking this process can result in cell cycle arrest, senescence, andapoptosis. Therefore, DNA replication forks are the targets of mostcancer chemotherapeutics, including agents that induce DNA lesions, suchas camptothecin (CPT) and cisplatin, plus those that stall forks, suchas gemcitabine and 5-fluorouracil. In addition, radiotherapy (RT), whichis used to treat ˜50% of all cancers, induces DNA damage. If notrepaired, this DNA damage may block or collapse DNA replication forksand kill cancer cells. However, a drawback of these therapies is thatthe cancer cell may become resistant to the radiation or chemotherapy.Reasons for resistance include increased tolerance for DNA lesions, andenhanced capacity for DNA damage response and repair. It has been shownthat that cancer cells that are resistant to RT or chemotherapeuticdrugs have abnormally high DNA repair capacity, and inhibition of DNArepair has successfully sensitized the cancer cells to cytotoxicity fromchemotherapeutic drugs.

One major conserved DNA repair enzyme is the DNA2 helicase/nuclease(DNA2). Complete inactivation of either the helicase or nucleaseactivity of DNA2 in cells from a wide range of organisms, includingyeast and humans, induces cell cycle arrest and cell death. Disruptionof DNA2 has been associated with human disease. A splice-site mutationthat causes decreased levels of human DNA2 gives rise to Seckelsyndrome, a primordial dwarfism syndrome. Other mutations are linked tobreast and gastric cancers. Interestingly, the DNA2-deficient Seckelcells show markers of senescence, where cells are viable but cease toproliferate.

DNA2 plays three key roles that allow the cancer cells to resist theintrinsic and extrinsic DNA replication stresses induced by chemotherapyor RT: flap removal during DNA replication, double-strand break (DSB)resection during repair and telomere replication and recombination, andstabilization and restart of reversed replication forks (Wanrooij andBurgers, 2015). During replication, DNA2 removes the long 5′ RNA/DNA“flaps” that arise during Okazaki fragment processing indifficult-to-replicate genomic regions. In yeast, ScDNA2 is probably themajor nuclease for RNA primer removal during Okazaki fragmentmaturation, in collaboration with flap endonuclease 1 (FEN1 or Rad27).For DSB repair, DNA2 acts in one of the two major DSB resectionpathways. DNA2 acts with the Bloom Syndrome (BLM) helicase or WernerSyndrome (WRN) helicase in end resection at a critical early step afterlicensing by limiting cleavage by the MRN/CtIP complex. BLM (or WRN),moving on the 3′ terminated strand, unwinds the duplex end to create a“fork”; DNA2 acts as a nuclease on the complementary strand and degradesthe 5′ end to produce 3′ ssDNA tails for strand invasion duringhomology-directed repair (HDR), BIR, and S phase checkpoint activation.This resection activity functions in parallel to and independently ofresection by exonuclease 1 (EXO1). At stalled replication forks, DNA2acts to stabilize, repair and restart forks to allow completion ofreplication. DNA2 also acts in signaling as both an activator and atarget of checkpoint kinases. For instance, DNA2 is required to directlyactivate the yeast master signaling kinase ATR. Furthermore, DNA2 is atarget of checkpoint effector kinase Rad53/Chk1/2, and is required toregulate potentially deleterious fork reversal and template switchingduring replication fork stalling in yeast and humans. DNA2 can also playa negative role when the RAD51, BRCA1, BRCA2 and the FA/BRCA (Fanconianemia/Breast cancer) pathway is impaired. Like MRE11, which functionsupstream of DNA2, DNA2 is involved in the excessive resection seen incells deficient in fork protection. Thus, DNA2 must be highly regulatedto protect genome stability.

Biochemically, DNA2 is well defined, though structure/function studieshave been limited by lack of a crystal structure (Pavletich, 2015,eLife). In particular, although it is known that the helicase andnuclease are coordinated, and the nuclease catalytic site and helicasemotifs are defined, the DNA binding sites remain elusive. Biochemicaland genetic experiments have demonstrated an intricate interactionbetween the nuclease and helicase (Bae et al., 2001; Budd and Campbell,2000, 2009; Kao et al., 2004a; Kao et al., 2004b; Levikova et al.,2013). Furthermore, biochemical studies indicate that there is a singlemajor DNA binding site interacting at the junction of the flap anddownstream duplex DNA that is required for both the nuclease andhelicase activities (Stewart et al., 2010). The motifs in the DNA2protein that govern this major binding site remains elusive. Finally,although mutations in the nuclease domain often also affect the helicaseactivity, no mutations in the helicase domain have been shown to reducethe nuclease activity significantly. Mutational analysis is typicallyused to study DNA binding, but we report the results of an alternativestrategy: the isolation of small molecule inhibitors of DNA2.

Elimination of the DNA2 gene sensitizes cells to radio- andchemo-therapeutic agents

We were interested in DNA2 as a target of inhibition because yeast andhuman cells that are depleted of DNA2 are sensitive to agents that causereplication stress, such as CPT and cisplatin (Budd & Campbell, 2000;Karanja et al, 2012; Peng et al, 2012). DNA2 is recruited toionizing-radiation (IR)-induced subnuclear foci in chicken and humancell lines (Hoa et al., 2015). To further investigate the biologicalroles of DNA2 in mammals, we established DNA2 knockout (dna2^(−/−))mouse ES (MES) cells. The dna2^(−/−) MES cells were viable, presumablydue to backup repair pathways, perhaps involving EXO1, as in yeast;although their proliferation rate was approximately 50% of the WT MEScells. To investigate if DNA2 knockout caused the cells to be moresensitive to DNA damaging agents, we treated WT and dna2^(−/−) MES cellswith γ-irradiation (IR) and CPT and observed that dna2^(−/−) MES cellswere significantly more sensitive than WT cells to both IR and CPT(FIGS. 8A-8B). Taken together with previous work (Duxin, J. P., Dao, B.,Martinsson, P., Rajala, N., Guittat, L., Campbell, J. L., Spelbrink, J.N., and Stewart, S. A. (2009). Human Dna2 is a nuclear and mitochondrialDNA maintenance protein. Molecular and cellular biology 29, 4274-4282.Duxin, J. P., Moore, H. R., Sidorova, J., Karanja, K., Honaker, Y., Dao,B., Piwnica-Worms, H., Campbell, J. L., Monnat, R. J., and Stewart, S.A. (2012); Karanja et al., 2012, 1014), these findings suggested thatDNA2 is a useful candidate for sensitizing cancer cells to DNAdamage-inducing therapeutic agents.

Virtual High Throughput Screening and Experimental Validation forInhibitors of DNA2

We employed a three-dimensional structural model for virtual screeningof small molecules that bind to DNA2. The homology model for the DNA2helicase domain was based on the crystal structures of the Upf1-RNA U15complex (PDB 2XZL (Chakrabarti et al., 2011)) and the human Upf1-ADPcomplex (PDB 2GK6 (Cheng et al., 2007)), which have high sequenceidentity with DNA2. Although the sequence identity (30%) between DNA2and Upf1 was at the lower limit for homology modeling, we were able tosuccessfully build the Upf1-based DNA2 model structure (FIG. 1A). Afteraligning the DNA2 helicase domain with UPF1, the homology model wasbuilt with the SWISS-MODEL tool and refined with the Schrodinger ProteinPreparation Wizard to fix missing residues and hydrogen positions (FIG.1A). We predicted druggable sites on the DNA2 model using anin-house-developed Druggable Site Prediction by FDA-approved drugs (DSP)methodology, which uses a diverse subset of 100 FDA-approved drugmolecules to dock around the protein surface and predict best bindingsites on the protein surface. The definition of the “best binding site”is based on the numbers of the tested drugs bound to the proteinpockets. We identified three docking pockets for screening, designatedas Sites 1, 2, and 3 with 53%, 24%, and 12% of the tested drugs bound tothem, respectively (FIG. 1A). Sites 1 and 3 are predicted to make closecontact with DNA and Site 2 is close to the ATP binding and hydrolysismotifs of the helicase domain. The proposed Site 3 is composed of thelimited residues conserved between DNA2 and UPF1 in the N-terminal modelsequence and interacts with the DNA and Site 1 (FIG. 1A). Moreimportantly, molecular dynamics simulation results further indicated theDNA2 model was stable and reasonable, making it a good choice for insilico screening of DNA2 small molecule inhibitors. Refinement of thestructure by molecular dynamics simulation showed that the site centersare stable and that the N-terminal domain stays as shown in FIG. 1B. Theaverage root-mean-square deviation (RMSD) of site centers was only 2-3 Åduring simulation, although the RMSD of some flexible loop regions couldbe more than 15 Å, which indicates that the predicted sites arestabilized to allow ligand binding. Based on an alignment of helicasefamily members, there are seven highly conserved helicase motifsincluding I, Ia, II, III, IV, V, and VI in DNA2. The I, II, and IVmotifs are for ATP binding and hydrolysis, whereas the Ia, III, and Vmotifs are the DNA binding domains in other helicases. Domains IV and VIcoordinate the DNA unwinding and hydrolysis. One predicted site of drugbinding, Site 1, contains amino acids in the Ia motif. Site 2 containsresidues in motif I, as well as in motifs V and VI (FIG. 1C). Thisinformation allowed us to focus our search for candidate DNA2 inhibitorson the most likely small molecule binding sites. It also guided us to apocket that might further illuminate how DNA2 interacts with its DNAsubstrate.

We then conducted a virtual high throughput screening (vHTS) formolecules binding to Site 1 because Site 1 gave the best FDA drug screenscore and is predicted to affect DNA binding, making it a favorabletarget site. Our model also suggested that Site 1, which interacts withSite 3, might be a shared component of both the helicase and nucleaseactivity and that inhibitors bound to Site 1 might clarify thecoordination of the nuclease and helicase activities of DNA2. We used anin-house-developed Multiple-Stage Full-Coverage (MSFC-VS) algorithm toscreen an in silico a library of 260,071 compounds from the NationalCancer Institute Developmental Therapeutics Program (NCI DTP) libraryfor binding to Site 1. This generated a list of 40 compounds (Table S1).

TABLE 1 Identification of the top 40 hits by virtual screening based onthe DNA2 3-D model DNA- NSC MW Docking Nuclease dependent No. No.*Formula (Dalton) Scores** inhibition ATPase C1 6284 C3H3N3O3 129.07−8.25 − − C2 7861 C6H6N4O2 166.14 −8.63 − − C3 9432 C7H8N4O 164.16 −9.25− − C4 12754 C5H8N2OS 144.19 −7.59 − − C5 15765 C10H6N2O5 234.17 −8.3 ++++ C6 20260 C9H15N2O15P3 484.14 −10.86 + + C7 33120 C18H18C12O6 401.24−7.34 − − C8 39858 C32H36N6O8S4 760.92 −7.41 ++ ++ C9 42753 C8H9F3O2194.15 −6.88 − − C10 55521 C9H6F6O 244.13 −7.05 − − C11 57727 C5H9N3O2143.13 −6.91 − − C12 64878 C7H7N3S 165.22 −7.04 − − C13 65634 C11H14N2O190.24 −7.71 − − C14 79004 C4H5N5S 155.18 −6.58 − − C15 79197 C4H9N3O3147.13 −9.24 − − C16 84922 C16H11NO3 265.26 −7.6 − − C17 85277C16H14N2O3 282.29 −10.06 − − C18 99439 C11H13N3O5 267.24 −11.61 − − C19102552 C6H9N3O2S 187.22 −9.28 − − C20 103797 C13H17N3O6 311.29 −11.05 −− C21 110391 C9H6N4O 186.17 −7.14 − − C22 115812 C16H12FNO3 285.27 −9.03− − C23 119749 C3H5N3O2 115.09 −8.03 − − C24 129784 C10H8N2O4 220.18−6.75 − − C25 140380 C19H29ClN6O6S 504.99 −7.44 − − C26 157740C12H13N5O4 291.26 −11.14 − − C27 166583 C6H4Cl2N4O 219.03 −7.77 − − C28170103 C10H3F7N2 284.13 −6.78 − − C29 211332 C5H11N5 141.17 −7.86 − −C30 266142 C6H6N2O3 154.12 −7.27 − − C31 291643 C9H10N2O6 242.19 −10.87− − C32 305488 C14H14N2O4 274.27 −9.26 − − C33 313976 C51H70N10O24P2S21340.00 −12.74 − − C34 321117 C10H8N2O2 188.18 −7.81 − − C35 329951C11H8N2O 184.19 −8.56 − − C36 360177 C6H6N4O2 166.14 −8.24 ++ ++ C37367734 C9H12N2O5S 260.27 −10.05 − − C38 375395 C8H4N2O6 224.13 −8.35 ++++ C40 382898 C30H18Cl3N15O7 806.92 −6.54 − − *NSC No.: the NCI'sinternal ID number. **Docking Score: Based on Schordinger Glide dockingsoftware at XP precision.

Although Site 1 is located in the putative helicase domain, we chose tosearch for inhibitors that might affect both the nuclease and helicaseactivities, because previous studies suggested that they may compete forthe same DNA binding site. We therefore screened these compoundsbiochemically for their inhibition of DNA2 nuclease activity usingpurified recombinant hDNA2 and a well-defined flap substrate. Among the40 identified compounds, 4 molecules inhibited the DNA2 nucleaseactivity (FIG. 1D and Table 1). Among these compounds,4-hydroxy-8-nitroquinoline-3-carboxylic acid, designated as compound C5,had the top Glide XP Docking Score (−8.3 kcal/mol), and displayed thehighest cytotoxicity to human cancer cells (FIG. 1E and Table 1). Todetermine if C5 specifically targets DNA2, we tested if C5 will inhibitthe enzyme activities of two similar structure specific nucleases, FEN1and EXO1. We found that C5 poorly inhibited the activities of the othertwo nucleases, in contrast to DNA2 (FIGS. 8C-F). Taken together, C5 is aspecific inhibitor of DNA2 nuclease activity in vitro.

Kinetic Analysis of Compound C5 in Inhibiting DNA2 Activity

In order to determine the IC50 of C5 for inhibition of the nucleaseactivity of DNA2, we conducted kinetic analyses. Using a flap DNAsubstrate, we first studied the time course of the nuclease activity inorder to determine the proper time interval for kinetic analysis atvarious substrate concentrations (FIG. 9A). We found that the enzymeactivity was linear at 1-10 minutes, and we conducted the assays in thisrange. To evaluate the mechanism of inhibition of nuclease activity byC5, we measured the nuclease activity of DNA2 at various concentrationsof inhibitor and substrate (FIG. 2A). Using a Lineweaver-Burk plot, weevaluated the competition patterns using competitive, noncompetitive,and uncompetitive models. The competitive inhibition model fits best tothe inhibition data with C5. To extract the intrinsic inhibitionconstant of C5 for DNA2, we obtained apparent inhibition constant, orIC50, values, for C5 at a series of substrate concentrations (FIG. 2B).Extrapolation of the observed IC50 values to limiting substrateconcentrations, as described in the Experimental Procedures section,gave an inhibition constant of 20 μM (FIG. 2C).

The kinetic studies suggested that C5 acts as a competitive inhibitor ofDNA2 nuclease activity. A competitive model predicts C5 binding to DNA2should block binding of DNA2 to the DNA substrate. Consistent with themodel, we found that C5 inhibited the ATPase activity of DNA2, which isdependent on DNA binding (FIGS. 2D and 2E). To further test this model,we evaluated DNA2 substrate binding in the presence of various C5concentrations directly, by electrophoretic mobility shift assay (EMSA).We found that DNA2 effectively bound flap DNA substrates, leading toreduced electrophoretic mobility; but the addition of C5 reduced theformation of the DNA2-substrate complex (FIGS. 2F and 2G). The inhibitorconcentration needed to reduce the DNA2-substrate complex formation to50% is 30 μM, which is comparable to the IC50 value for inhibition ofthe DNA2 nuclease activity. Finally, we tested the ability of C5 toinhibit DNA2 helicase activity. Using the DNA2 helicase substrate inwhich an M13 phage DNA is hybridized to a 5′ tailed oligonucleotide(Masuda-Sasa et al., 2006), we found that C5 inhibits the 5′ to 3′,end-dependent DNA helicase activity of DNA2 (FIG. 9B, compare lanes 3and 6, 4 and 7, 5 and 8) (Balakrishnan et al., 2010; Masuda-Sasa et al.,2006). Based on our results, we suggest that although our homologystructure does not show the nuclease domain, that contacts in bothATPase and helicase domains are required for nuclease activity,consistent with the recent X-ray crystal structure of murine DNA2 (80%identical to human DNA2).

Validation of Site 1 on DNA2 as the Binding Pocket for Compound C5 andas a DNA Binding Motif

C5 was identified by virtual screening for small molecules that bind toa pocket (Site 1) near the putative DNA binding site in ourcomputational DNA2 model. Based on the 3-D model, we searched theresidues within 6 Å spheres around the predicted C5 binding site. Weidentified multiple residues, including V682, L686, F696, L697, R698,L699, G700, L729, L732, Y733, Q736, Q738, V739, and T741, that may playa key role in coordinating C5 binding (FIG. 3A). To test if C5 indeedbinds to the pocket at Site 1, we substituted these residues withalanine. The mutant and WT DNA2 proteins were purified and the nucleaseactivities were assayed. Although Site 1 lies in the helicase domain,many of the mutations altered the nuclease activity in the absence ofinhibitor, presumably because the Site 1 pocket is close to the putativeDNA substrate binding pocket. These are the first mutations within thehelicase domain that have been demonstrated to concomitantly affect thenuclease activity. Other mutants, however, maintained intact nucleaseactivity (FIGS. 10A and 10B). We chose F696A and L732A with enzymeactivity similar to the WT to test their sensitivity to compound C5.Both the nuclease activity and DNA binding activity of the two mutantswere less sensitive to C5 than wild type (FIGS. 3B and 3C and FIG. 10C).The compound C5 IC50s for nuclease activities and DNA substrate bindingwere greater than 250 μM for F696A and L732A, compared to 30 μM for theWT. This suggests that the mutations impair the interaction of C5 withthe designated binding site (Site 1) of DNA2 and that this site doesdefine a crucial flap substrate DNA binding domain.

C5 Displays On-Target Effects on DNA2

Next, we determined the cytotoxicity of DNA2 inhibitor C5 and evaluatedwhether the compound had on-target effects on DNA2 in cultured cells. Wehave measured IC50 values of C5 with a panel of 18 cell lines of 4 majortypes of cancers by a cell proliferation assay (Chou 2010). The IC50values among the different cell lines varied from 7 μM to 70 μM, whichis comparable to the estimated enzymatic IC50 value of 20 μM (FIG. 4A).Furthermore, we considered that if the toxic effects of C5 were due totargeting DNA2, then cells lacking DNA2 should be resistant to theeffects of compound C5. As anticipated, we found a reduced spontaneoussurvival rate in human and mouse cells with either shRNA-mediatedknockdown (FIG. 4B) or knockout (FIG. 4C) of DNA2. We found that WT MEScells and human MEF7 cancer cells treated with C5 showed a 60% survivalrate, compared to the untreated WT control (FIGS. 4B and 4C), a resultsimilar to the effects of DNA2 knockdown and knockout. Importantly, wefound that treatment of dna2^(−/−) MES or DNA2 knockdown human cancercells with C5 did not further reduce the survival rate, suggesting thatthe cytotoxic effects of compound C5 were primarily due to specificeffects on DNA2 at the given concentration. This suggests that due toresidual viability, those normal cells may be less sensitive toinhibitors than cancer cells, with repair and checkpoint defects. Thisalso suggests that other enzymes, such as FEN1 or EXO1, cannotcompletely compensate for the loss of DNA2, indicating that DNA2inhibitors are likely to have significant physiological effects incancer cells. The results suggest that C5 is a potent and specificinhibitor of DNA2 and that virtual docking is a valid method to helpidentify DNA2-specific drugs.

In yeast, pathways mediated by DNA2 overlap with repair or replicationby FEN1, EXO1, or MRE11 and not with others (Budd, M. E., Tong, A. H.,Polaczek, P., Peng, X., Boone, C., and Campbell, J. L. (2005). A networkof multi-tasking proteins at the DNA replication fork preserves genomestability. PLoS genetics 1, e61). Thus, mutants defective in FEN1, EXO1,or MRE11 are viable only in the presence of active DNA2. Double mutantsdeficient in DNA2 and FEN1, EXO1, or MRE11, respectively, are inviable,especially in the presence of DNA damage. This effect is calledsynthetic lethality. To test for synthetic lethality, deficiency inDNA2, can be induced either by genetic mutation or, if a specificinhibitor is available, by inhibition of DNA2. We reasoned that if DNA2is the major cellular target of C5, then cells deficient in FEN1, EXO1,or MRE11 should be hypersensitive to C5, i.e. C5 should synergize withdeficiency in any one of these three genes. We have now shown that thisis the case in both yeast and in human cells (FIGS. 13A-13C, human cellsand FIG. 14, yeast cells). FIGS. 13A-13C show that siRNA knockdowns ofFEN1, EXO1, or MRE11 are killed by increasing levels of C5, while thecells treated with scrambled siRNA, are resistant. To control for theefficacy of C5, we took advantage of a cell line carrying a conditionalgenetic knockout of DNA2, HCT116^(flox/−/−) (Karanja, K. K., Lee, E. H.,Hendrickson, E. A., and Campbell, J. L. (2014) Cell Cycle 13,1540-1550). After floxing, i.e. induction of Cre nuclease withtamoxifen, this strain becomes a complete DNA2 knockout. As shown inFIGS. 13A, 13B, and 13C, gray curves, the knockout of DNA2 issynthetically lethal with deficiency in any of the three genes and thelevel of sensitivity to 10 μM C5 is similar to the level of lethalityseen in the DNA2^(−/−/−). (The Mre11 result is also described in Liu etal EBioMedicine 6 (2016) 73-86). Both inhibition of DNA2 by C5 andgenetic knockout of DNA2 with tamoxifen produces synthetic lethalitywith FEN1, EXO1 or MRE11 deficiency.

Yeast contains many genes encoding transporters that preventaccumulation of drugs to toxic levels. Fortunately, these genes arecoordinately regulated by Pdr1 (pleiotropic drug resistance regulator).When Pdr1 is fused to gene encoding a strong repressor, Pdr1 binds tomany resistance genes and the repressor turns off expression, allowingaccumulation of inhibitors. The chimeric repressor is expressed on aplasmid and can be easily transferred to many genetic backgrounds(Nitiss and Wang, 1988; Stepanov et al., 2008). This has improved yeastas a model for testing therapeutic agents in mechanism based-drugdiscovery.

We have created a number of drug-sensitive yeast strains that allow usto test the efficacy of DNA2 inhibitors in ways that are highly likelyto measure only on-target effects of the compounds (Stepanov, A.,Nitiss, K. C., Neale, G., and Nitiss, J. L. (2008); Mol Pharmacol 74,423-431). In particular, rad27 mutants, lacking FEN1, specifically needDNA2 to survive in the presence of damage. FEN1 is not inhibited by C5(Liu et al EBioMedicine 6 (2016) 73-86). So we tested if the inhibitorincreased the sensitivity of rad27 to DNA damage. As shown in Fig. N+1,DNA2i C5 synergizes with MMS and with deletion of rad27, mimicking theexact and specific behavior of dna2 mutants (Budd, M. E., and Campbell,J. L. (1997) Mol Cell Biol 17, 2136-2142). We further show thatderivative 2 of C5 (also referred to herein as C5-2, shown below) ismore effective than C5. The compound C5-2 has the formula:

As seen in FIG. 14, this is a dilution series of yeast, and in thepresence of inhibitor, rad27 is much more sensitive to MMS and to hightemperature than in the absence. Wild-type is not affected by theselevels of C5 drugs. This shows that the inhibitor functions in yeast andthat its target is likely DNA2.

We previously showed that knockdown of DNA2 suppressed the cisplatinsensitivity of FANCD2−/− fibroblasts (Karanja, K. K., Lee, E. H.,Hendrickson, E. A., and Campbell, J. L. (2014) Cell Cycle 13,1540-1550). This suggests that C5 should also suppress the cisplatinsensitivity of FANCD2−/− cells, which might protect non-cancerous cellsin FA patients from treatment of tumors with cisplatin (see FIG. 15).Cells were treated with cisplatin in the presence of increasing amountsof C5 or in the absence of C5. Survival was determined as described inKaranja et al (Karanja, K. K., Lee, E. H., Hendrickson, E. A., andCampbell, J. L. (2014) Cell Cycle 13, 1540-1550). The experiment wasperformed in duplicate. As shown in FIG. 15, at 125 nM cisplatin, cellstreated with 10 μM C5 showed an increase in survival compared to cellsnot treated with C5.

C5 suppresses DNA DSB repair end resection, over-resection of nascentDNA in cells defective in fork protection, and restart of stalled DNAreplication forks

To further validate C5 as a DNA2 inhibitor, we tested its effects onDNA2 functions known to be affected by knockdown or deletion of DNA2 inprevious studies (Howard et al., 2015; Karanja et al., 2014; Karanja etal., 2012; Peng et al., 2012). We first determined the effect of C5 ontwo recombination pathways, SSA (single-strand annealing) and homologousrecombination (HR), using I-SceI/GFP-based reporter assays. Since thesepathways are most active in S/G2 (FIG. 11A), we determined GFP positivecells on the G2 population. We found that both SSA and HR were reducedby C5 in a dose-dependent manner (FIG. 5A). For comparison, at 60 μM C5,SSA and HR were reduced to the same level as in an siRNA DNA2 knockdowncarried out in parallel (FIG. 11B).

We next wanted to verify if the defects in recombination assays were dueto inhibition of end resection. During the early steps of recombination,DNA2 in complex with BLM or WRN protein resects DSB ends, producingssDNA 3′ overhangs (Karanja et al., 2012; Nimonkar et al., 2011;Sturzenegger et al., 2014). The ssDNA overhangs are coated with RPA,which is then phosphorylated by ATR (Zou and Elledge, 2003). To measureresection, we determined the level of phosphorylated RPA2 (S33 or S4/8)in cells treated with C5 in the presence or absence of CPT. CPTstabilizes cleavable complex intermediates in topoisomerase I reactionswhich collapse into DSBs when encountered by a replication fork (Hsianget al., 1989; Patel et al., 2012). CPT increased phosphorylated RPA2(P-RPA), as measured on western blots and by immunofluorescence ofP-RPA2 foci (FIGS. 5B-E). C5 significantly reduced the CPT-induced P-RPAlevel in γH2AX-positive cells (FIGS. 5B-E). The level of C5 used herereduced RPA phoshporylation to the same extent as we observed inparallel studies using siRNA DNA2 knockdown (compare FIGS. 5C-5E and5F-5H). Interestingly, we also noted that C5 alone caused backgroundincrease in γH2AX (FIGS. 5B-5H), presumably because the DNA2 inhibitoritself causes replication stress, similarly as shown previously forshDNA2 knockdown (Duxin, J. P., Dao, B., Martinsson, P., Rajala, N.,Guittat, L., Campbell, J. L., Spelbrink, J. N., and Stewart, S. A.(2009). Human Dna2 is a nuclear and mitochondrial DNA maintenanceprotein. Molecular and cellular biology 29, 4274-4282. Duxin, J. P.,Moore, H. R., Sidorova, J., Karanja, K., Honaker, Y., Dao, B.,Piwnica-Worms, H., Campbell, J. L., Monnat, R. J., and Stewart, S. A.(2012); Karanja et al., 2012).

When DNA replication forks are stalled in S-phase, DNA2 plays animportant role in stabilizing the stalled forks, in preventing DSBformation, and in resection of the nascent strand to prepare for therestart of replication (Hu et al., 2012; Karanja et al., 2012; Thangavelet al., 2015; Weitao et al., 2003b; Weitao et al., 2003d). A recentstudy using DNA fiber assays indicated that knockdown of DNA2 by siRNAsinhibits replication fork restart in HU-treated cells or cells treatedwith low levels of CPT (Thangavel et al., 2015). We have conducted DNAfiber assays to evaluate the effect of C5 on restart of forks treatedwith low levels of CPT, which in contrast to high CPT levels does notcause DSBs but results in increased positive supercoiling and forkslowing and stalling (Ray Chaudhuri et al., 2012), or with HU, whichinhibits production of nucleotide precursors. In absence of C5, 80% or75% of replication forks could restart (red-green tracts) in HU-treatedor CPT-treated cells, respectively (FIG. 6A). However, 20 μM C5 reducedthe percentage of restarting forks to 60% and 50% in the HU-treated orCPT-treated cells, respectively (FIG. 6A). This level of inhibition at20 μM C5 was equivalent to knockdown of DNA2 using siRNA (FIG. 6B).These results extend previous studies on DNA2 functions duringreplication restart (Ray Chaudhuri et al., 2012; Teicher, 2008) and showthat DNA2 is more sensitive to inhibition at stalled replication forksthan at a single DSB (FIGS. 5A-5H). C5 also inhibited restart in cellstreated with high levels of CPT, resulting in replication associatedDSBs (FIG. 12A).

Fork protection is a DNA break repair-independent pathway suppressinggenomic instability (Schlacher et al, 2011; Schlacher et al, 2012). Thefork protection pathway is mediated by BRCA2, BRCA1, RAD51, members ofthe Fanconi anemia pathway, and BOD1l. In the absence of any of thesefactors, excessive nascent DNA degradation occurs at stalled replicationforks. This degradation is prevented if the end-resection protein MRE11is inhibited either by siRNA knockdown or by inhibition of MRE11nuclease by the small molecule mirin (Schlacher et al., 2011; Schlacheret al., 2012). MRE11 functions upstream of DNA2 in resection ofdouble-strand breaks, and DNA2, in addition to MRE11 has been implicatedin over-resection at stalled replication forks in BOD1L and in RAD51impaired cells, since over-resection is suppressed by knockdown of DNA2in the absence of BOD1L or in a RAD51 mutant cell line (Higgs et al.,2015; Wang et al., 2015). To determine if C5, like DNA2 knockdown,suppressed nascent DNA degradation and resulting accumulation of ssDNAat stalled forks, we monitored phospho-RPA (P-RPA) foci in BRCA2- orBOD1L-depleted or mock transfected U2OS cells with and without HUtreatment (FIGS. 6C and 6D). In the absence of HU, in mock-depletedcells we saw few cells with greater than 15 P-RPA foci/per cell, andneither mirin nor C5 significantly decreased the number of P-RPApositive cells, consistent with the fork protection pathway being intact(FIG. 6C). After treatment of the BRCA2- or BOD1L-depleted U2OS cellswith HU, which stalls forks but is not expected to produce DSBs(Petermann et al., 2010; Schlacher et al., 2011; Schlacher et al.,2012), and which are fork protection defective, we observed a dramaticincrease in P-RPA positive cells indicative of nascent DNA degradationupon treatment with HU (FIG. 6C and FIG. 12B). This degradation wassuppressed by C5. The potency of C5 was estimated by comparing theeffect of mirin, the MRE11 inhibitor, in the same experiment.Remarkably, 20 μM C5 has a comparable potency to 50 μM mirin in reducingthis degradation (FIGS. 6C-6D). To exclude off-target effects of mirinin our experiments, we showed that another potent MRE11 inhibitor, PFM39reduced P-RPA foci to the same extent as mirin in the BRCA2-deficientcells (FIG. 6D). Finally, we showed in a parallel experiment that thelevel of reduction in P-RPA foci caused by C5 in BRCA2-deficient cellsis also equivalent to that observed in a DNA2 knockdown (FIG. 6D andFIG. 12B). Our results are in keeping with previous work showing thatknockdown of DNA2 counteracts excessive nascent strand degradation inboth BOD1L and in RAD51 impaired cells (Higgs et al., 2015; Wang et al.,2015). Importantly, extending previous reports, we show that MRE11 andDNA2 both are responsible for degrading stalled replication forks infork protection defective cells, including BOD1L and BRCA2. We concludethat C5 suppresses the fork protection defect of the BRCA2- orBOD1L-deficient cells by inhibition of DNA2. Taken together, the datashows that C5 is a specific inhibitor of DNA2 activities at stalledreplication forks in vivo.

C5 Sensitizes Cells to PARP Inhibitors

To test our hypothesis that inhibition of DNA2 synergizes with otherchemotherapeutic agents, we treated cells with CPT and C5. We found thetwo agents to be synergistic for cellular lethality (FIG. 7A). Thesedata uncover the potential for C5 in increasing killing efficiencies ofDNA damaging chemotherapeutics.

PARPs have been shown to play an important role in DNA single-strandbreak (SSB) repair and at stalled replication forks (Bryant et al.,2009; Hu et al., 2014; Yang et al., 2004; Ying et al., 2012), andinhibition of PARPs resulted in accumulation of SSBs and consequent DSBsin the cells (Fisher et al., 2007; Okano et al., 2003). More recently,PARPs have also been implicated in loading DNA damage response proteinsto DSB sites (Hu et al., 2014; Li and Yu, 2013). Because DNA2 appears tofunction in many of the same pathways as the BRCA breast cancer genes,including DSB repair and replication fork protection, and PARPinhibition shows synthetic lethality with BRCA1 or BRCA2 mutants(Fathers et al., 2012; Fong et al., 2008), we considered that PARPinhibition might increase the effectiveness of the DNA2 inhibitor. Totest this hypothesis, we determined the survival of MCF7 breast cancercells cultured in the medium containing compound C5 and/or a MK4827, aPARP inhibitor (Jones et al., 2009). We found that MK4827 and C5 had astrong synergistic effect in inhibiting the survival of MCF7 cells(FIGS. 7B and 7C). The IC50 for MK4827 and C5 was 0.8 μM and 1.9 μM. Thecombination index for MK4827 (1 μM) and C5 (2 μM) was 0.13, indicating avery strong synergy between two drugs. These data support a model whereDNA2 and PARP collaboratively participate or have complementary roles inDNA damage response, DSB repair, BER repair pathways, and replicationfork protection, which will be an important issue to dissect in futurestudies.

Discussion

We have used a partial DNA2 protein structure based on the homologybetween the helicase domain of DNA2 and yeast Upf1-RNA U15 complex andhuman Upf1-ADP complex (Chakrabarti et al., 2011; Cheng et al., 2007) toidentify 3 well defined pockets (Sites 1, 2, and 3), where drug likemolecules can preferentially dock. We then used a virtual screenconsisting of docking of 260,721 NCI deposited small molecules to thesesites to identify DNA2 inhibitors. This screen is similar to a previousvirtual screen used to identify inhibitors of ribonucleotide reductase(Chen et al., 2015; Zhou et al., 2013). We characterized one inhibitor,C5, which we demonstrate biochemically inhibits nuclease, DNA dependentATPase, helicase, and DNA binding activities of DNA2. Through a seriesof functional analyses, we have pinpointed the specific functions ofDNA2 that C5 targets to explain its cellular toxicity. C5 suppressesreplication-coupled DSB end resection and restart of either HU- orCPT-stalled DNA replication forks. C5 also inhibits over-resection ofnascent DNA in cells defective in replication fork protection, such asBRCA2. All these data support our conclusion that virtual screening canbe efficient, and that C5 is a promising lead compound to developsensitizers for cancer chemotherapeutics that cause replication stress.

It is interesting to note that C5, which our model and data suggestbinds to the helicase domain of DNA2, can suppress the nucleaseactivity. We propose that this occurs because C5 can reduce DNAsubstrate binding to a site in the helicase domain necessary to activatethe nuclease. The dramatic effect of the inhibitor on the nucleaseactivity, its predicted binding site, and more importantly, the effectof mutations we identified in helicase domain 1A on the nucleaseactivity and DNA binding reveal that the DNA binding site in helicasedomain 1A (counterintuitively) is indeed critical for nuclease activity.This in turn suggests that DNA contacts in the nuclease site are notsufficiently strong to promote nuclease activity; multiple domains ofDNA2 have to interact with DNA to elicit nuclease activity. These majornew insights into the structure/function mechanism of this importantclass of enzyme, fused helicase/nuclease (including AddAB and RecB),extend recent reports on the X-ray crystal structure of murine DNA2 (80%identity to human DNA2 in the helicase domain)(Zhou et al., 2015). Thestructure shows DNA threading through a tunnel in the enzyme, firstcontacting the nuclease. The helicase domain follows and binding occursas domain 1A and then 2A contact the DNA in the tunnel (Zhou et al.,2015). This explains why both nuclease and helicase utilize threadingmechanisms (Bae et al., 2002; Balakrishnan et al., 2010; Kao et al.,2004a). Our work in turn provides direct evidence for requirement forboth these domains for nuclease and helicase activity. Taken togetherwith previous biochemical studies (Bae et al., 2002; Balakrishnan etal., 2010; Kao et al., 2004a; Stewart et al., 2010), a “thread, bind,and cleave” model now best explains how DNA2 nuclease works. C5 couldeither block threading, so that domain 1A does not come into contactwith DNA or C5 could block binding to 1A or 2A. While we know about theactive sites, the inhibitor will be useful in studying the role of thehelicase, which is still conjectural. Co-crystals of DNA2 with C5 shouldbe very informative with respect to the mechanism we propose for C5function based on the putative binding to Site 1 in our homologystructure. The putative common DNA binding site, predicted by ourresults and shown in the crystal structure, explains how the nucleaseand helicase compete for the same substrate, as proposed by Levikova etal (Levikova et al., 2013) and that the nuclease catalytic site contactsmust be disrupted for helicase to be active when duplex DNA isencountered. Therefore, in addition to possible therapeutic uses, the C5inhibitor and well-designed derivatives will be valuable in futurestudies of how the helicase and nuclease activities are co-regulated andintegrated.

This study of C5 adds cogent support to more circumstantial evidencethat has accumulated that a major, and not minor, function of DNA2 bothin yeast and human is to participate in the protection, remodeling andrestart of stalled replication forks. We first reported this when weshowed, using 2D gels, that yeast dna2-1 mutants led to replication forkcollapse or remodelling into intermediates thought to be chi-structured(recombination) intermediates or reversed forks and DSBs, whenreplication forks stalled at the endogenous FOB1 protein-mediatedreplication fork barrier in the rDNA (Weitao et al., 2003a; Weitao etal., 2003c). In S. pombe, furthermore, it was shown that DNA2 was actedon by the checkpoint to prevent fork collapse upon stalling at a similarbarrier (Hu et al., 2012). More recently, human DNA2 has been shown inelegant DNA spreading experiments using knockdowns to be crucial forrestart of forks stalled by CPT and specifically to promote limitedresection necessary for restart (Thangavel et al., 2015). This activitymust be tightly controlled, however, because the restoration of forkprotection by inhibition of DNA2 in both BRCA2- and BOD1L depletedcells, at very low levels of the DNA2 inhibitor C5 (20 μM) and to thesame extent as knockdown of DNA2 (FIGS. 6A-6D) if not properlyregulated, over-resection by DNA2 can lead to excessive fork degradationand genome instability (Higgs et al., 2015; Wang et al., 2015). Thiscoincides with the demonstration that depletion of DNA2 can suppress thecisplatin sensitivity of FANCD2^(−/−) cells. By contrast to thesereplication functions, we found that higher levels of C5 were requiredto achieve the same level of inhibition as by knockdown of DNA2 (FIGS.5A-5H) when measuring SSA and HR in the GFP reporter assays, which arenot thought to depend on on-going replication,

Until this study, there were no known small molecule inhibitors of DNA2.Discovering and testing additional DNA2 inhibitors will not only beinvaluable for characterizing the integrated DNA2 enzymatic activitiesbut will also enhance the preparation of advanced inhibitors foranticancer regimens, either alone or in combination with otherchemotherapeutics. Importantly, chemical inhibition of DNA2 displayedcytotoxicity to DNA2-proficient mouse and human cells but not towarddna2^(−/−) MES DNA2-deficient cells of human cells after DNA2 knockdown,supporting the assertion that we have found a DNA2-specific inhibitor.The use of inhibitors plus mutations to elucidate the mechanism of DNA2nuclease/helicase activation as a basis for understanding its regulationin vivo is critical to design new therapeutic regimens. Inhibitors allowone to monitor the acute response of cells to the absence of DNA2, whichis an important distinction from genetic studies, whose interpretationis always made difficult when studying essential genes such as DNA2. Inthe current study, we tested if targeting DNA2 may exploit a specificvulnerability in the cancer cells, assuming that normal cells are betterprotected by intact checkpoints and redundant repair processes.

We also tested the possible potentiation of PARP inhibitors by DNA2inhibitor C5, because previous work suggested that DNA2 and PARP mightparticipate in overlapping repair and replication stress responsepathways (Bryant et al., 2009; Wanrooij and Burgers, 2015; Yang et al.,2004; Ying et al., 2012). PARP inhibitors have proved promising intreating BRCA-deficient tumors but fall short in that many such tumorsare resistant. We tested if DNA2 might be partially responsible byperforming a complementary function using the C5 DNA2 inhibitor. Indeed,C5 potentiated PARPi and vice versa. This “synthetic lethality” suggestsnovel approaches to applications of PARPi therapy. It also increasesconfidence that DNA2 and PARP function in response to replication stressthat might occur in response to oncogene activation or treatment withDNA damaging agents such as CPT. Our demonstration of the stimulation ofPARP inhibition by C5 may suggest that DNA2 plays a complementary rolewith PARP at stalled replication forks, where PARP has been shown tomediate replication fork restart in conjunction with MRE11, a nucleasethat acts upstream of DNA2 in resection functions (Bryant et al., 2009;Shibata et al., 2014). In sum, the data presented here show that DNA2inhibitors sensitize cancer cells to DNA damaging agents and additionalagents used in current therapy and therefore may be feasible anti-canceragents.

DNA2 is required for efficient telomere replication and repair. This isone way in which C5 and derivatives thereof may work. Additionally, DNA2is also involved in telomere homeostasis, both in resection and inOkazaki fragment processing. Since 85% of tumor cells regeneratetelomerase, there is some interest in telomerase inhibitors to fightcancer. An inhibitor of DNA2, such as for example the compoundsdescribed herein, might counteract the tumor's ability to make telomeresand therefore survive.

The generation of 3′ G strand overhangs at telomere ends may play a rolein regulating telomerase action and occurs by still unclear mechanisms.We show by an inducible short telomere assay that Sae2 and the Sgs1 RecQhelicase control two distinct but partially complementary pathways fornucleolytic processing of S. cerevisiae telomeres, with Sae2 functionrequiring its serine 267 phosphorylation. No processing activity isdetectable in sae2Delta sgs1Delta cells, while the Exo1 exonucleasecontributes to telomere end processing and elongation in both sae2Deltaand sgs1Delta cells, suggesting that Exo1 telomeric function requireseither Sgs1 or Sae2 action. Moreover, Dna2 might also support Sgs1activity, as it acts redundantly with Exo1, but not with Sgs1. Finally,both length maintenance and G strand overhang generation at nativetelomeres are affected in sae2Delta sgs1Delta cells, further supportingthe notion that Sae2 and Sgs1 combined activities control telomerelength by regulating telomere processing.

Experimental Methodology

Protein purification and nuclease activity assay. All WT and mutant DNA2proteins in this study were expressed as 3× Flag-tagged recombinantproteins in 293T cells and were purified using affinity chromatographyas previously described (Ronchi et al., 2013). The nuclease assay wasconducted as previously described (Zheng et al., 2008).

ATPase assays and EMSA assays. The ATPase assay was conducted aspreviously described (Masuda-Sasa et al., 2006). WT DNA2 nucleasecleaves the DNA substrate, which makes it technically difficult todisplay the helicase and ATPase activities. We therefore chose to usethe D294A mutant, which is defective in nuclease activity but hassimilar ATPase activity to WT DNA2, to test the inhibitory effects of C5(Masuda-Sasa et al., 2006). The EMSA was performed as describedpreviously (Hellman and Fried, 2007).

Inhibition mode and nonlinear regression to determine the inhibitor Ki.For different concentrations of substrate, we increased the enzymeconcentration in the same ratio to obtain the precise relative meanvelocity, using 0.5 nM, 1.5 nM, 2.5 nM, 5 nM DNA2 for 5 nM, 15 nM, 25nM, 50 nM substrates, respectively. For analysis of competitiveinhibition, the Lineweaver-Burk plot was used with variousconcentrations of substrate and inhibitor. At a given substrateconcentration, apparent inhibition constants of the inhibitor(IC50_(observ)) were derived from fit of the data to Eq 1,

$\begin{matrix}{{{fraction}\mspace{14mu}{inhibition}} = \frac{\lbrack I\rbrack}{{{IC}\; 50_{obsev}} + \lbrack I\rbrack}} & (1)\end{matrix}$

The inhibition constant K_(i), which describes inhibitor binding to DNA2in the absence of substrates, is related to observed IC₅₀ values byequation (2),

$\begin{matrix}{{{IC}\; 50_{obsev}} = {K_{i}\left( {1 + \frac{\lbrack S\rbrack}{K_{M}}} \right)}} & (2)\end{matrix}$in which K_(M) is the Michaelis constant for the substrate. At limitingsubstrate concentrations, the IC50_(observ) values approaches K_(i).Thus, the K_(i) value is obtained from extrapolation of the[S]-dependence of IC50_(observ).

Cell culture, measurement of IC50, clonogenic assay, and cellproliferation assay. Cancerous and non-cancerous cells were culturedbased on the culture instructions from the American Type CultureCollection (ATCC). Chemical compounds for the candidate DNA2 inhibitorswere requested from NCI DTP (http://dtp.nci.nih.gov/). CPT was purchasedfrom Sigma (St. Louis, Mo., purity >99%) and MK4827 was from MedChemExpress (Monmouth Junction, N.J.). The cell survival rate of MES orhuman cancer cells under different treatments was measured by clonogenicassays following a published protocol (Franken et al, 2006). Briefly,500 cells were seeded in a 6-well plate and incubated in culture mediumcontaining DMSO or drugs, fresh medium with or without C5 for thecultured cells was changed every 3-4 days. For clonogenic assay, theplate was washed with PBS buffer after 14 days of culture and thecolonies were fixed and stained with crystal violet solution and thenumber of colonies (>50 cells) was counted. The combination index,indicating the synergistic effect of the compounds, was measuredfollowing a previously published method (Chou, 2010). To measure theIC50 with the proliferation assay, 1,000-2,000 cells were seeded in a96-well plate and incubated in culture medium containing DMSO or drugs,fresh medium with or without C5 for the cultured cells was changed everythree days. After six days, the CellTiter 96 One Solution Reagent(Promega, Madison, Wis.) was added to the culture medium to measure theviable cells and the absorbance at 490 nm (A490), which is positivelycorrelated to the number of cells, in each well was measured. Therelative number of viable cells in the untreated control group wasarbitrarily set as 1, and the relative number of viable cells in aC5-treated well was calculated by dividing its A490 by that of thecontrol well.

Immunofluorescence staining. Cells (on cover-slips) with or withoutvarious drug treatments for 24 h were fixed with 4% paraformaldehyde,permeabilized with 0.1% Triton X100, blocked with the Image iT FX signalenhancer (Invitrogen), and incubated (1.5 h, room temperature) with theindicated primary antibodies. The antibodies against γ-H2AX andphosphorylated RPA2 were from Sigma Aldrich. The cells were then washedwith PBS buffer and incubated (1 h, room temperature) with thecorresponding secondary antibodies (1:200, Invitrogen). The slides werethen washed with PBS buffer, counter stained with DAPI and analyzed witha fluorescence microscope (Olympus AX70).

DSB repair reporter assays. HDR-GFP and SSA-GFP were integrated in U2OScells (Gunn and Stark, 2012; Howard et al., 2015). The U2OS cells weretransfected with the I-sceI expression vector, or the empty vector andGFP vector as negative and positive controls, respectively. At 3 hoursafter transfection, we changed the culture medium to fresh medium withor without compound C5, and cultured the cells for 3 more days. Thecells were harvested and the GFP+ frequencies (repair frequencies) weredetermined by flow cytometry using a CyAn ADP Analyzer (Beckman Coulter,Inc.).

Establishment of DNA2 knockout ES cells. To construct the knockoutvector for the mouse DNA2 allele, two DNA fragments corresponding to themouse DNA2 gene were inserted into the poly-linker A and B,respectively, on the gene targeting vector, PKO scrambler NTK(Invitrogen, Carlsbad, Calif.) containing a neomycin or puromycinselection marker. One DNA fragment (5′ arm) covered DNA sequences fromintron 1 to intron 3 of the mouse DNA2 gene, and the other DNA fragment(3′ arm) corresponded to DNA sequences from intron 7 to intron 12 of themouse DNA2 gene. The knockout vector (neomycin) was electroporated intoembryonic stem cells of the 129S1 genetic background. Recombinationbetween the knockout vector and the WT DNA2 allele resulted in a mutantDNA2 allele, which deletes the exons 4-7 and disrupts the mouse DNA2gene. DNA2+/−ES cells were selected by neomycin marker and confirmedwith Southern blotting analysis. A second DNA2 knockout vector(puromycin) was electroporated into the DNA2+/−ES cells and DNA2−/− EScells were selected by both neomycin and puromycin markers and confirmedwith Southern blotting and PCR analysis

DNA fiber assay. DNA fibers performed and the data were analyzed asdescribed previously (Schwab & Niedzwiedz, 2011; Techer et al, 2013;Thangavel et al, 2015). Briefly, A549 cells with or without DNA2inhibition by C5 or siDNA2 were pre-incubated with 50 μM IdU for 40 min,left untreated or treated with indicated drugs in IdU medium, and thenincubated with 250 μM CldU for 40 min. The cells were then collected andsuspended in ice-cold PBS at 7.5×10⁵ cells/ml. The labeled cells (2 μl)were dropped on a glass slide and waited for 5 min to partially dry, andthen lysed with 7 μl of lysis buffer (0.5% SDS in 200 mM Tris-HCl, pH7.4, and 50 mM EDTA) for 2 min. The slides were tilted ˜15° to allow DNAfiber spreads. After drying, the slides were fixed in 3:1methanol/acetic acid for 10 min. The DNA was then denatured in 2.5 M HClfor 80 min, followed by PBS washes for 15 min (triple), and the slideswere blocked with 5% BSA for 30 min. The slides were then incubated with1:50 rat anti-BrdU (Abcam, clone BU1/75 (ICR1), for detection of CldU)followed by 1:50 mouse anti-BrdU (BD Biosciences, clone B44, fordetection of IdU) for 1 hour. After incubation with the primaryantibodies and extensive washes, slides were incubated with 1:200 AlexaFluor 488-conjugated anti-rat (Life Technologies) and Alexa Fluor 555anti-mouse (Life Technologies) for 1 h. Next, the slides were mountedwith ProLong Gold Antifade reagent (Life Technologies). Images weretaken with a Zeiss AxioCam 506 Mono microscope.

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of treating cancer in a patient in needthereof, the method comprising administering to the patient atherapeutically effective amount of a compound, a pharmaceuticallyacceptable salt thereof, a tautomer thereof, or a pharmaceuticallyacceptable salt of a tautomer thereof; wherein the cancer is breastcancer, colon cancer, prostate cancer, lung cancer, or ovarian cancer;and wherein the compound is:


2. The method of claim 1, further comprising administering to thepatient a chemotherapeutic agent.
 3. The method of claim 2, wherein thechemotherapeutic agent is a PARP inhibitor.
 4. The method of claim 2,wherein the chemotherapeutic agent is a topoisomerase inhibitor.
 5. Themethod of claim 4, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.
 6. The method of claim 2, wherein thechemotherapeutic agent is irinotecan, topotecan, camptothecin,lamellarin D, etoposide, teniposide, doxorubicin, daunorubicin,mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, HU-331,epigallocatechin gallate, genistein, quercetin, resveratrol, or acombination of two or more thereof.
 7. The method of claim 1, furthercomprising administering radiation to the patient.
 8. The method ofclaim 2, further comprising administering radiation to the patient.